Methods for identifying and isolating antigen-specific T cells

ABSTRACT

The present invention is directed to methods for the identification and isolation of antigen-specific T cells using a novel class of artificial antigen presenting cells. The resulting T cells may be used to produce expanded T cell populations as well as for modulating T cell responses. In general, the artificial antigen presenting cells useful in such methods are liposomes that contain MHC:peptide complexes presented on the outer surface of the liposome. Such artificial antigen presenting cells may also include accessory molecules, co-stimulatory molecules, adhesion molecules, and other molecules irrelevant to T cell binding or modulation that are used in the binding of artificial antigen presenting cells to solid support systems that may be used in the retrieval and identification of antigen-specific T cells.

RELATED APPLICATIONS

This application claims priority to provisional application No.60/105,018, filed Oct. 20, 1998.

GOVERNMENT SUPPORT

This invention was made with government support under NIH Grant Nos.AR40770, AI37232, and AR41897. The government has certain rights in thisinvention.

FIELD

The field of this invention is immunology, specifically, methods ofpreparing artificial antigen presenting cells and their application tomethods of isolating antigen-specific T cells, methods of modulating theT cell response and methods of treating conditions which would benefitfrom the modulation of the T cell response, for example, transplantationtherapy, autoimmune disorders, allergies, cancers and viral andbacterial infections.

BACKGROUND

The following description provides a summary of information relevant tothe present invention. It is not an admission that any of theinformation provided herein is prior art to the presently claimedinvention, nor that any of the publications specifically or implicitlyreferenced are prior art to the invention.

The immunologic arts have advanced markedly over the past ten years. Thecomplexity of the science explaining aspects of the field is immense. Weset forth below in this section a discussion concerning known aspects ofvarious elements involved in immunogenic responses and concepts in theart that are related to the invention disclosed herein.

T Cells

T lymphocytes (i.e., T cells) are part of the immune system, whichdefends the body against bacterial, viral and protozoal infection, aswell as aberrant molecules that contain epitopes recognized as non-self.The recognition of non-self molecules as well as the destruction ofinfectious agents carrying non-self antigens is a function of T cells.These cells provide for the cell-mediated immune responses of adaptiveimmunity.

Infecting pathogens are generally accessible to extracellular antibodiesfound in the blood and the extracellular spaces. However, some infectingagents, and all viruses, replicate inside cells where they are notexposed to, and cannot be detected by, extracellular antibodies. Inorder for these foreign agents to be accessible to the cell-mediatedimmune response, the cells harboring such pathogens must either“express” antigenic motifs of the infecting agents on the surface of thecells or the antigenic motifs must be shed from-the-cells, e.g. by celldeath, to be accessible to and subsequently expressed on the cellmembrane of phagocytic antigen presenting cells (“APCs”) thatparticipate in the immune process.

Antigens derived from replicating virus for example, are displayed onthe surface of infected cells where they may be recognized by“cytotoxic” T cells which may then control the infection by recognizingthe viral antigen and killing the cell. The actions of such cytotoxic Tcells depends upon direct interaction between the antigenic motif of theinfecting agent expressed on the surface of the infected cells and the Tcell's receptors having a specificity for the motif.

Although T cells are important in the control of intracellularinfections, some foreign agents evade such control because theyreplicate only in the vesicles of macrophages; an important example isMycobacterium tuberculosis, the pathogen that causes tuberculosis.Whereas bacteria entering macrophages are usually destroyed in thelysosomes, which contain a variety of enzymes and bactericidalsubstances, infectious agents such as M. tuberculosis, survive becausethe vesicles they occupy cannot fuse with the lysosomes. The immunesystem provides for fighting such agents by a second type of T cell,known as a T helper cell, which helps to activate macrophages and inducethe fusion of lysosomes with the vesicles containing the infectingagents. The helper cells also bring about the stimulation of otherimmune mechanisms of the phagocyte. T helper cells may further beinvolved in initiating and/or sustaining the immune system's release ofsoluble factors that attract macrophages and other professional APCs tothe site of infection.

Additionally, specialized “helper” T cells play a central part in thedestruction of extracellular pathogens by interacting with B cells.Depending on the type of infection being controlled, participating Thelper cells may have an inflammatory or Th1-like phenotype, or asuppressive Th2-like phenotype.

T Cell Receptors

T cell receptors (TCRs) are closely related to antibody molecules instructure and are involved in antigen binding. Variability in theantigen binding site of the TCR is created in a fashion similar toantibodies in that a large capacity for diversity is available. Thediversity is found in the CDR3 loops of TCR variable regions which arefound in the center of the antigen-binding site of the TCR. Thediversity that is obtainable by TCRs for specific antigens is alsodirectly related to an MHC molecule on the APC's surface to which theantigenic motif is bound and presented to the TCR.

One type of MHC that is involved in presenting processed antigen isclass II MHC. The antigen or peptide binding site for a peptide on aclass II MHC molecule lies in a cleft between the alpha and beta chainhelices of the MHC molecule. In another type of MHC, the class I MHC,the binding site for a peptide lies in a cleft between the two alphahelices of the alpha chain. From the arrangement of highly variableantigens complexing with MHC molecule alleles, it is understandable thatthe mechanism of TCR recognition involves a combined distribution ofvariability in the TCR which must correlate with a distribution ofvariability in the ligand (i.e., antigen/MHC molecule complex).(Garboczi, et al., Nature Vol. 384; 134–41; Ward and Quadri, Curr OpImmunol. Vol. 9:97–106; Garcia, et al., Science, Vol. 279:1166–72).

MHC Molecules

In general, T cell responses to non-self motifs depend on theinteractions of the T cells with other cells containing proteinsrecognized as non-self. In the case of cytotoxic T cells and Th1 cells,non-self proteins (i.e. antigens) are recognized on the surface of thetarget cell (such as an infected cell). Th2 cells, on the other hand,recognize and interact with antigen presented by professional antigenpresenting cells such as dendritic cells, and B cells. Dendritic cellsnon-specifically internalize antigen while B cells bind and internalizeforeign antigens via their surface immunoglobulin. In any case, T cellsrecognize their targets by detecting non-self antigenic motifs (e.g.,peptide fragments derived from for example, a bacterium or virus) thatare expressed either on infected cells or other immune cells, e.g.phagocytic APC. The molecules that associate with these peptide orantigen fragments and present them to T cells are membrane glycoproteinsencoded by a cluster of genes bearing the cumbersome name “majorhistocompatibility complex” (MHC). These glycoproteins were firstidentified in mice in studies examining the effects on the immuneresponse to transplanted tissues. In humans, the MHC equivalent has beentermed HLA for “human leukocyte antigen”. In general, the term MHC isused to describe generally the molecules in the mammalian immune systeminvolved in the presentation of antigenic motifs to T cells. As usedspecifically in this Letters Patent, MHC means any majorhistocompatibility complex molecule, either class I or class II, fromany mammalian organism including a human, such molecule comprisingfull-length MHC molecules or sub-units thereof further comprising MHCencoded antigen-presenting glycoproteins having the capacity to bind apeptide representing a fragment of an autoantigen or other non-antigenicor antigenic sequence (e.g., a peptide), said MHC further having anamino acid sequence that is expressed and purified from natural sources,or by any artificial means in prokaryotic or eukaryotic systems havingdifferent glycosilations, or of either natural or synthetic origin thatcontains or comprises a modification of a natural MHC sequence.

The actions of T cells depend on their ability to recognize antigenicmotifs on cells (such as cells harboring pathogens or that haveinternalized pathogen-derived products). T cells recognize peptidefragments (e.g., pathogen-derived proteins) in the form of complexesbetween such peptides and MHC molecules that are expressed on thesurface of “antigen presenting cells”.

The two types of MHC molecules, i.e., MHC class I and MHC class II,deliver peptides from different sources (class I being intracellular,and class II being extracellular) to the surface of the infected cell.The two classes of MHC molecules vary with respect to the length ofpeptides that they are able to present. The binding pocket of the MHCclass I molecules is blocked at either end, thereby imposing severerestrictions on the size of peptides it can accommodate (8–10 residues).The binding groove of the MHC class II molecules on the other handallows peptides to protrude from the ends, and consequently much longerpeptides (8–30 residues) can bind. (Rudensky, et al. Nature, Vol.353:622–27; Miyazaki, et al., Cell, Vol. 84:531–41; Zhong, et al., J.Exp. Med., Vol. 284:2061–66).

Antigen Processing

Peptides bound to MHC class I molecules are recognized by CD8+ T cells(cytotoxic T cells), and those bound to MHC class II molecules arerecognized by CD4+ T cells (helper T cells). Two functional subsets of Tcells are thereby activated to initiate the destruction of antigenicmotifs, and thereby the source (e.g. a pathogen) which may reside indifferent cellular compartments. CD4+ T cells may also help to activateB cells that have internalized specific antigen, and in turn give riseto the stimulation of antibody production against the antigenic motifsof the extracellular pathogens.

Infectious or antigenic agents can reside in either of two distinctintracellular compartments. Viruses and certain bacteria replicate inthe cytosol or in the contiguous nuclear compartment, while manypathogenic bacteria and some eukaryotic parasites replicate in theendosomes and lysosomes that form part of the vesicular system. Theimmune system has different strategies for eliminating such agents fromthese two sites. Cells containing viruses or bacteria located in thecytosol are eliminated by cytotoxic T cells which express thecell-surface molecule CD8. The function of CD8 T cells is to killinfected cells.

Immunogenic agents located in the vesicular compartments of cells (whichmay or may not have been involved in the internalization ofextracellular matter) are detected by a different class of T cell,distinguished by surface expression of the molecule CD4. CD4 T cells arespecialized to activate/modulate other cells and fall into twofunctional classes: Th1 cells which activate various immune competentcells to have the intravesicular non-self antigenic agents they harbordestroyed, and Th2 cells which help to activate B cells to, among otherthings, make antibody against such foreign agents.

To produce an appropriate response to infectious microorganisms, T cellsneed to be able to distinguish between self and foreign or non-selfmaterial coming from the different processing pathways. This is achievedthrough delivery of peptides to the cell surface from each of theseintracellular compartments by the different classes of MHC molecules. Asnoted above, MHC class I molecules deliver peptides originating in thecytosol to the cell surface, where the antigen (i.e., non-selfrecognized peptide) is expressed in association with the MHC molecules(antigen:MHC complex) and is recognized by CD8 T cells. Likewise, MHCclass II molecules deliver the non-self peptides originating fromextracellular sources to the cell surface, where they are recognized byCD4 T cells.

Antigen Presenting Cells

When naïve T cells encounter for the first time a specific antigen onthe surface of an antigen-presenting cell (APC), they are activated toproliferate and differentiate into cells capable of contributing to theremoval of the antigen and its source (e.g. an infecting pathogen). TheAPCs are specialized in that they express surface molecules thatsynergize with a specific antigen in the activation of naïve T cells.APCs become concentrated in the peripheral lymphoid organs, to whichthey migrate after trapping antigen while circulating in the periphery.APCs present peptide fragments or antigenic motifs to recirculatingnaïve T cells. Arguably, the most important APCs are dendritic cellswhose known function includes the presentation of antigen to Macrophagesand are important in phagocytosis of cells that provide a first line ofdefense against infecting agents. APCs are also known to be activated byarmed effector T cells. B cells also serve as APCs under somecircumstances.

One of the features of APCs is the expression of co-stimulatorymolecules including B7-1 and B7-2 molecules. Naïve T cells will respondto an antigenic motif only when the same APC presents to the T cell boththe specific motif recognized by the TCR and a B7 molecule which isrecognized by CD28 or CTLA-4, the receptors for B7 existing on the Tcell surface. (Anderson, et al., J. Immunol., Vol. 159:4:1669–75). Theactivation of T cells by APCs leads to proliferation of the activated Tcells and to the differentiation of their progeny into armed effector Tcells. The proliferation and differentiation of T cells depends on theproduction of cytokines (such as the T cell growth factor, IL-2) andtheir binding to high-affinity receptors on the activated T cell. Tcells whose TCRs are bound to antigens in the absence of co-stimulatorymolecules fail to make cytokines and instead become anergic. This dualrequirement for both receptor/antigenic interaction and co-stimulationhelps to further mediate naïve T cell response.

Proliferating T cells develop into armed effector T cells, the criticalevent in most adaptive immune response. Once an expanded clone of Tcells achieves effector function, the T cell clone progeny can act onany target cell that displays or expresses a specific antigen on itssurface. Effector T cells can mediate a variety of functions. Thekilling of infected cells by CD8 cytotoxic T cells and the activation ofprofessional APC by Th1 cells together make up cell-mediated immunity.The activation of B cells by both Th2 and Th1 cells help to producedifferent types of antibodies, thus driving the humoral immune response.(Kirberg, et al., J. Exp. Med., Vol. 186:8:1269–75).

T Cell Activation

T cells generally become sensitized to antigens by becoming trapped inlymphoid organs as the T cells drain into lymph nodes through which theycirculate. Antigens introduced directly into the bloodstream, or thatreach the bloodstream from an infected lymph node, are picked up by APCsin the spleen for example where lymphoid cell sensitization occurs inthe splenic white pulp. The trapping of antigen by APCs that migrate tothese lymphoid tissues combined with the continuous recirculation of Tcells through the tissues ensures that rare antigen-specific T cellswill encounter their specific antigen being presented by an APC.

The recirculation of naïve T cells through the lymphoid organs isorchestrated by adhesive interactions between lymphocytes andendothelial cells. Naïve T cells enter the lymphoid organs through aprocess which is thought to occur in a number of steps. The first stepin this process is mediated by selectins expressed on the T cell. Forexample, L-selectin on naïve T cells binds to sulfated carbohydrates onthe vascular addressins GlyCAM-1 and CD34. CD34 is expressed onendothelial cells in many tissues but is properly glycosylated forL-selectin binding only on the high endothelial venule cells of lymphnodes. L-selectin binding promotes a rolling interaction, which iscritical to the selectivity of naïve lymphocyte homing. Although thisinteraction is too weak to promote extravasation, it is essential forthe initiation of the stronger interactions that then follow between theT cell and the high endothelium, which are mediated by molecules with arelatively broad tissue distribution. (Finger, et al., Nature, Vol.379:266–9).

Stimulation by locally bound chemokines activates the adhesion moleculeLFA-1 on the T cell, increasing its affinity for ICAM-2, which isexpressed constitutively on all endothelial cells, and ICAM-1, which, inthe absence of inflammation, is expressed only on the high endothelialvenule cells of peripheral lymphoid tissues. The binding of LFA-1 to4ts4igands, ICAM-1 and ICAM-2 plays a major role in T cell adhesion toand migration through the wall of the blood vessel into the lymph nodes.Bachmann et al., Immunity, Vol. 7:549–57).

The high endothelial venules are located in the lymph nodes. This areais inhabited by dendritic cells, which have recently migrated from theperiphery. The migrating T cells scan the surface of these APCs forspecific antigen:MHC complexes. If they do not recognize antigenpresented by these cells, they eventually leave the node via an efferentlymphatic vessel, which returns them to the blood so that they canrecirculate through other lymph nodes. Rarely, a naïve T cell recognizesits specific antigen:MHC complex on the surface of an APC, which thensignals the activation of LFA-1, causing the T cell to adhere stronglyto the APC. Binding to the antigen:MHC complex also activates the cellto proliferate and differentiate, resulting in the production of armed,antigen-specific T cells. The number of T cells that interact with eachAPC in lymph nodes is very high, as can be seen by the rapid trapping ofantigen-specific T cells in a single lymph node containing antigen.

Identification and Isolation of Antigen-Specific T Cells

As noted above, T cells represent a major component of the body's immunedefenses against bacterial, viral and protozoal infections, as well asnon-self antigenic motifs from other sources. T cells have also beenimplicated in the rejection of cancerous cells. Autoimmune disordershave also been linked to antigen-specific T cell attack against variousparts of the body. One of the major problems hampering the understandingof and intervention on the mechanisms involved in these disorders is thedifficulty in identifying T cells specific for the antigen to bestudied. Accordingly, it is of great interest to be able to identifyantigen-specific T cells. Additionally, it would be of great therapeuticbenefit if T cells specific for a particular antigen could be (i)enriched and then reintroduced in a disease situation, (ii) selectivelydepleted in the case of an autoimmune disorder, or (iii) modified toalter their functional and/or phenotypic characteristics. Thus,identification and isolation of antigen specific T cells is an essentialrequirement in immunology and medicine to understand and modulate immuneresponses.

Identification of antigen-specific T cell populations is generallyaccomplished by indirect means in animal models, such as by evaluatingmembrane markers correlated to activation or maturation of these cells.The majority of these studies were performed in transgenic systems(Ignatowicz, et al., Cell, 84:521–29; Sebzda et al., Science, Vol.263:1615–18; Jameson et al., Ann. Rev. Immunol., Vol. 13:93–126).Analysis is generally done by means of flow cytometry, where a detectoron a machine is capable of identifying cells bound to fluorescentsubstrates, such as fluorescent antibodies. Positively identified cellscan be sorted for further use. Quantitation and isolation ofantigen-specific T cells is usually accomplished by limiting dilutionand cloning techniques. When using sorted cells, these approaches becomequite cumbersome and are sometimes inaccurate, since the biologicaleffects of antigen recognition can spread beyond the cells recognizingthe antigen. For instance, upon engagement of the specific MHC:antigenicpeptide complex, T cells produce cytokines that can affect expression ofthe same markers of activation in non-specific bystander T cells. Hence,in order to isolate and characterize cells with specificity for a givenantigen, alternative procedures, such as T cell cloning, need to beapplied. These techniques often require many months of technicalprocedures before results can be obtained. The rate of success, inparticular for human systems, is quite low, and the population selectedmay not necessarily represent the biologically relevant component of theimmune response to a given peptide. The direct interaction of a specificT cell with the antigen:MHC complex would thus be a preferred basis forT cell isolation.

Theory of the Invention

The immunoregulation art has advanced steadily in recent years. Thescientific literature contains many studies showing interactions andmodulation effects between specific molecules and cell types. However,no discovery has been presented that is able to apply the knowledge thathas been gained by the extensive research in the field toward a methodor device that can be used in a comprehensive package for carrying outthe identification, isolation, and modulation of immunoregulatory cellsfor the purpose of advancing the ultimate goal of such knowledge, i.e.improved treatment regimens for various states of disease.

We have discovered a platform technology for advancing treatmentregimens requiring the immunoregulation of immune cells that centersaround the use of an artificial antigen presenting cell (APC). Thisplatform technology may be designed or programmed on demand for use inthe treatment of a broad spectrum of specific disease states. Moreover,this system is versatile and applicable to all situations where theisolation, identification, and modulation of T cells is of clinicalimport. We have recognized the relevance of several types of molecularentities to the stimulation/activation and modulation response of Tcells in their role within the immune system and have incorporated theseentities into artificial APCs. We use such artificial APCs to captureand manipulate antigen specific T cells.

Historically, programming and using T cells therapeutically has beenhampered by the problem of finding a means by which the cells can behandled for such manipulation and observation of the effectiveness ofthe manipulation applied. We have solved this problem by adopting thetheory that a T cell can best be manipulated by using APC likestructures and encorporating into such structures molecules constructedto (1) bind the “artificial” APC to specific T cell types, (2) stimulateor modulate only specifically bound T cells for any desired response,and (3) bind the artificial APC to a solid support in situations whereanchoring the APC to a specific location is desired.

Prior to our invention, no comprehensive system has been disclosed, norwas it obvious that such a system would function as desired, to achievea platform that is universally applicable to activating and modulation Tcells. As can be seen by the numerous following distinctions, much ofthe art has centered only on basic research relating to molecules andtheir association with T cell response.

Distinctions

Kendrick et al., U.S. Pat. No. 5,595,881 (the '881 disclosure) discuss amethod for the detection and isolation of MHC:antigen-restricted T cellswhich is performed by preparing the MHC:antigen complex, which complexis isolated by using metal chelating technology. The complexes thenbound to a planar solid support {i.e., a glass coverslip), followed inturn by combining the immobilized complex with a biological sample sothat the MHC:antigen complex may bind to and retain antigen-specific Tcells. Determination of the presence of reactive MHC:antigen complexesis carried out by observation of cell proliferation.

The method described in the '881 disclosure differs from the currentinvention in a number of substantial structural and functional ways.First, the MHC component of the complexes in the '881 disclosure areimmobilized on a solid support. The MHC component of the currentinvention is not bound to a solid support but is freely “floating”within the bilayer of a polysome membrane comprising aphosphotidylcholine and cholesterol component. The difference issubstantial in that the MHC:antigen complex of the '881 disclosure isnot able to participate in the migration or concentration of suchcomplexes in “capping” which is important to improved binding andactivation of bound T cells. Second, the '881 method is only directed tothe detection of the presence of “natural” APCs that are specific forpre-selected antigen-specific T cells after such T cells have beenisolated. The isolation of antigen-specific T cells is carried out byfirst performing a series of steps including binding antigen via a metalchelating process to a solid support, capturing on to the antigen MHCthe components that are antigen-specific, then isolating the MHC:antigencomplexes which are in turn bound to a planar solid support via alinker.

The current invention is also much more versatile. It is not concernedwith detecting natural APCs but is instead directed to the isolation andmanipulation of antigen-specific T cells. The manipulation of such Tcells is carried out for numerous applications such as directlyimpacting T cell function by modulating the T cell response. Themanipulation can be performed in either a column format, with means forsupporting the artificial APCs, and/or in free solution via flowcytometry (FACS). The current invention is able to modulate T cellfunction because the artificial APCs may be designed to specification inthat various functional molecules are incorporated into the APC thatactivate specific T cell responses. For example, in one embodiment ofthe current invention, known MHC molecules may be incorporated intoliposomes along with a labeled antigenic peptide for which such MHC hasspecificity (e.g., in the case of FACS a biotinylated antigen). Theliposome:MHC:biotinylated antigen complex may be used to bind toantigen-specific T cells and the fact of binding can be visualized byFACS followed by the sorting of the bound cells. Thus, no cellproliferation is necessary to identify and isolate antigen-specific Tcells.

In addition to the MHC:antigen complex, the “artificial APCs” used tocapture the antigen-specific T cells include accessory molecules to helpstabilize the MHC:antigen:TCR interaction, and may also includefunctional molecules such as co-stimulatory molecules which in oneembodiment may be used to activate T cells, adhesion molecules which maybe used to bind cells destined for a certain area of the body, and otheraccessory or functional molecules such as cytokines or antibodies tocytokine receptors, which are known to have immunomodulatory effectsupon T cells. Moreover, the current invention further provides forproper orientation of each of these molecules within the artificial APCmembrane by a novel use of an anchoring mechanism comprising GM-1 andthe β subunit of cholera toxin. In this aspect, the molecule of interestmay be connected to the cholera toxin subunit as a fusion protein or byuse of a linking moiety. By attaching the cholera toxin subunit to themolecule of interest, the cholera toxin may be bound by the GM-1 that isincorporated into and has affinity for the nonpolar region of theartificial APC membrane.

All of these molecules are incorporated into the liposomes of theartificial APCs in a free floating format. Other molecules may beincluded that do not influence the modulation of T cell response such asproteins that may be used to anchor the artificial APC to a solidsupport. Such molecules may also be produced as fusion proteins forproper orientation. As used herein such molecules that are notassociated with modulation or T cell binding are termed “irrelevant”molecules.

Additionally, a label may be attached to the antigen, the irrelevantmolecule, or the liposome component. Moreover, label may also benoncovalently associated within the lipids of the liposome.

The designs of these artificial APCs also allow for optional expansionexperimentation of T cell populations responding to the MHC: antigencomplexes associated in the cell like liposomes using a solution based(e.g., roller bottle) cell culture. The concept of the current inventionrepresents a substantial and heretofore unrecognized advance in theMHC:antigen complex T cell binding art in that the artificial APC (e.g.the example comprising liposome:MHC:antigen:accessorymolecule-functional molecule complex) is not restricted to complexes ofMHC:antigen alone or to a planar surface as is the case with much of theprior art. The importance of the structural differences cannot be overemphasized. The addition of the accessory molecules, as well asco-stimulatory molecules, and other proteins in proper orientation inthe liposomes of the current invention allow for substantially improvedbinding association and manipulation of T cells which is very importantin the identification and stimulation of antigen-specific T cells. Thisis especially true in solution based FACS analysis where functionalityof the antigen-specific T cells can be interpreted directly. Forexample, prior studies (Watts, T. H. Annals of the New York Academy ofSciences. 81:7564–7568.) respecting the modulation of T cells may beerroneous. There, it was demonstrated that planar membranes containingpurified MHC loaded with antigen fused to glass cover slips elicitedIL-2 production by T cells through the interaction of the T cell withthe MHC:antigen complex. It was also shown that the same complex whenformed in unilamellar vesicles (i.e., liposomes) elicited no response.Contrary to such teaching, we have found that liposome vesiclescontaining MHC: antigen complexes can in fact elicit strong responsewhen combined with accessory molecules such as LFA-1, and othermolecules such as co-stimulatory and adhesion molecules. We based ourtheory that liposomes could function without use of a planar array onthe observation (by the same study cited immediately above) that crudemembrane preparations of cellular material from which the MHC waspurified were effective in eliciting T cell responses in both planar andvesicular forms. Subsequently, we have discovered that “extraneous”matter existing in cell extracts that might be hypothesized to impartfunctionality to vesicular forms of lipid bilayers (as opposed tounilamellar liposomes alone) are not important to T cell binding andresponse. Rather, T cell binding and response is possible usingvesicular forms of liposomes containing specific molecules applied incombination with lipsomes (e.g., accessory molecules, co-stimulatorymolecules, and adhesion molecules).

Prior research has also been inconclusive respecting the use of MHCmolecules. For example, it has been shown (Buus, S. Cell. 47:1071–1077.)that a particular antigenic peptide binds solely to the alpha chain ofthe class II MHC IA^(d) molecule while other investigations have shownthat binding interactions between T cell receptors and MHC:antigenternary complexes use whole MHC, not just single chains of the MHC, todetermine peptide sequence motifs. Exactly how much of a MHC: antigencomplex must be presented is not absolutely known and may vary with Tcell specificity. We have directed our invention to the use of eitherwhole MHC molecules or those parts of the a and β subunits of Class Iand Class II MHC necessary for forming antigen binding cleft regions inthe binding of antigen peptides.

The current invention's use of co-stimulatory, adhesion and otheraccessory molecules in a “free floating” format also helps to bothanchor and direct the interaction between MHC:antigen:accessory moleculeand T cell receptors by providing a means by which T cells in the samplewill be presented with a structure more similar to that found in thenatural state. Specifically, the MHC:antigen:accessory moleculecomplexes in conjunction with other functional molecules are able tomigrate in proper orientation in the lipid bilayer of the liposomebecause of the use of a unique combination of lipids and surfactantmolecules, namely an optimal ratio of phosphotidylcholine andcholesterol respectively, included in the liposome matrix. These provideparticular protein presentation characteristics and easy proteinmigration properties to the surface of the liposome structure so thatthe MHC:antigen complexes can easily migrate to T cell binding locisimilar to “capping” events seen in natural APCs. Moreover, as shown inthe figures, the structure of our artificial APC liposomes allows forspecific “capping” of the liposomes on the surface of the T cells towhich the liposomes are bound. Additionally, interaction between the Tcell and artificial APC-associated molecules is further enhanced by themolecules being oriented in the lipid membrane such that their activesites are positioned facing outward on the APC. Without suchorientation, the ratio of properly oriented molecules to improperlyoriented molecules is around 50:50. This ratio is greatly increasedusing MHC, functional and accessory proteins that have attached thereto(either by fusion protein construction or by use of a linker) a choleraβ toxin subunit moiety which is placed in relation to the active centerof the protein of interest such that upon the β subunit being bound byGM-1 which is incorporated into the lipid layer of the artificial APC,the protein of interest will lay in the APC with the active site facingoutward.

Additional versatility is available with the current invention in thatthe artificial APCs may incorporate irrelevant molecules to be used inconjunction with separate solid support-based capture moieties forcapturing generic target motifs such as irrelevant molecules. Because ofthe capacity for the functional molecules to migrate in the liposome,the irrelevant molecules may be nonspecifically directed away from thebinding position of the T cells thus avoiding steric hindrances.Additionally, the system avoids a need for manufacturing specializedsolid phase capture substrates for each antigen-specific complex.

With regard to the capture of the APC by the solid phase component ofthe invention, we refer to target molecules used in the artificial APCfor binding to capture molecules of the solid support as “irrelevant”molecules because they do not impact the APC: T cell interaction. Such adesign further preserves the ability of the other molecules insertedinto the liposome to move freely and accommodate any capping of the Tcell's activation related molecules.

It has been recognized that the number of receptors on a T cell isvariable (Rothenberg, E. Science. 273:78–79.). It is also known that thenumber of TCRs and combination of co-stimulatory molecules and accessorymolecules varies with the maturation of the T cell (Dubey, C. J.Immunol. 157:3820–3289.). How many such receptors are needed in allsituations to elicit a T cell response is unknown. Moreover, it is knownthat presence of a co-stimulatory signal decreases the number ofreceptors necessary to activate a T cell (Viola, A. and Lanzavecchia, A.Science. 273:104–106.). We have provided for the uncertainties presentedby such data by providing a system that allows control over the numberof MHC:antigen:accessory molecule complexes relative to other functionalmolecules such as co-stimulatory, and adhesion molecules. The bindingand modulation of the T cell response at different stages of cellmaturation may be “fine tuned” using our invention.

In another system, Nag et al. in U.S. Pat. No. 5,734,023 (the '023disclosure), disclosed MHC subunits which were complexed with antigenicpeptides and “effector” molecules wherein such complexes were used toidentify T cell populations that were associated with autoimmunediseases. The complexes were used to destroy and anergize such T cellpopulations from a patient's blood cell population.

The effector molecules are described as such things as toxins,radiolabels, etc. which may be conjugated to the MHC or antigen portionof the complexes and which may effecutate the identification, removal,anergy, or death of such T cell populations. Such effector molecules arenot related to the attractive binding interactions or T cell responsesto effectuate a phenotype change in the cells. They are merely designedand intended to aid in the recognition and/or destruction of specific Tcell populations. Additionally, the '023 disclosure uses lipids in theconstruction of micelles which are designed for intravenous injection astherapeutics. The use of negatively charged acidic phospholipids (suchas phosphatidylserine) and the lack of cholesterol or GM-1 and choleratoxin subunit in the design of such micelles differs from that of thecurrent invention in substantial ways. For example, our invention usesneutrally charged phospholipids such as phosphotidylcholine (Pc). Wehave found that the design of our artificial APCs substantiallyincreases stability because of the Pc and cholesterol in environmentswhere IL-1 is present. IL-1 is known to interact with chargedphospholipids and destabilize liposome structure. Likewise, inenvironments where TNF is present, the permeability of liposomescomprised of charged phospholipids (e.g., phosphotidylserine) is greatlyaffected. In the same manner, environments where RNase is present mayalso affect charged phospholipid liposome structures. We have avoidedthe disruptive effects caused by molecules that are often present inmedia from which T cells are isolated by designing artificial APCs usingneutral phospholipids.

Additionally, the use of liposomes and the parameters associated withmicelle construction that are disclosed in the '023 disclosure arewholly associated only with the stability of MHC:antigen:effectormolecule complexes in the in vivo circulatory environment. There is norelation inherent or otherwise to the current invention, nor is thereinsight disclosed as to liposome construction containing co-stimulatoryand adhesion molecules or protein orientation mechanisms such as thebinding of cholera toxin by GM-1, or fused or linked moieties to theMHC, functional or accessory proteins of interest. Further, the '023disclosure does not discuss use of its micelle construction in thecontext of use of a MHC: antigen complex ex vivo where manipulation of Tcell function and the binding attraction between T cells and MHC:antigen complexes with respect to the current invention is of import.Moreover, the current invention does not use the technology disclosed inthe '023 disclosure of single chain MHC in liposomes. In contrast, in apreferred embodiment, our invention uses either whole MHC molecules orthose portions of the α and β subunits necessary to bind antigens andthat may be designed to have substantially favorable liposomestabilizing characteristics as well as binding capabilities when in thepresence of other functional molecules in the artificial APC asdisclosed herein.

In yet another recent disclosure, Spack et al. in U.S. Pat. No.5,750,356 (the '356 disclosure) describe a method for monitoring T cellreactivity using a modified ELISPOT assay which detects various factorsproduced by the stimulation of T cells with numerous factors in thepresence of natural antigen presenting cells. The current invention isdistinguishable from the '356 disclosed method in that the currentinvention uses artificial antigen presenting cells which haveincorporated therein various accessory, co-stimulatory, adhesion,cytokine, and chemokine molecules that provide substantial effect in thebinding and modulation of T cell responses. Additionally, in embodimentsthat require solid support binding, our APC includes irrelevantmolecules. Moreover, in another embodiment, our invention includesmechanisms to properly orient proteins of interest in the lipidmembrane.

In still another disclosure, Wilson et al. in U.S. Pat. No. 5,776,487disclose a use of liposome structures for determining analyte in a testsample wherein the liposome contains only an analyte specific ligand anda haptenated component used to bind to a receptor moiety on a solidphase. This combination allows for capturing a test analyte onto a solidsupport for detection. Thus, it is vastly divergent from the concept ofthe current invention.

Our methods and artificial APCs are further distinguished from otherrecent disclosures. For example, Altman et al., in U.S. Pat. No.5,635,363 (the '363 disclosure), discuss a method for labeling T cellsaccording to the specificity of their antigen receptor by preparing a“stable multimeric complex” comprised of four or more MHC moleculeshaving a substantially homogenous bound peptide population. Themultimeric antigen:MHC complex was said to form a stable structure thatbecause of its “stable multimeric” design, purportedly increases theaffinity of a T cell receptor for its specific antigen thereby allowingfor the labeling, identification and separation of T cells. Althoughsuch multimeric MHC components are known to bind T cells, they are notincorporated into liposome structures. Thus, the MHC complexes areunable to participate in capping type concentration. Moreover, the '363method does not use accessory, co-stimulatory, adhesion, or othermolecules to assist T cell binding and/or activation or modulation.

The current invention is further distinguishable over prior disclosuresin that our invention is based on the recognition that the valency ofthe liposome: MHC structure is multiple, and empirically determined.Moreover, we have provided for greater specificity in following APC:Tcell interaction due to one embodiment of our invention wherein theantigen is labeled rather than the MHC component (e.g. a biotinylatedantigen with a streptavidin molecule conjugated to a fluorochrome).

In light of the above noted distinctions, the disclosed artificial APCand use of a separate solid support containing a binding protein to bindirrelevant molecules on the artificial APC represents an especiallynotable improvement over prior art. For example, in the above mentionedprior technologies the design of complexes are such that simultaneousbinding and capping of the MHC: antigen and TCR/CD3/accessory moleculescannot occur. Capping is the phenomenon by which the T cell focuses therelevant molecules to the portion of the cell where binding hasoccurred, thus amplifying the binding, and subsequently the signaling ofthe event to the cell's other components. The current invention providesa specifically designed lipid bilayer similar to that of a natural cellwhich allows protein molecules, such as the MHC:antigen complexes tofloat freely, thus enabling the complexes to conform to any cappingevents the T cell may undergo. The consequence is a greater ability ofthe current invention to bind to, stimulate, and modulate T cells ondemand.

SUMMARY OF THE INVENTION

The present invention is directed to novel methods of isolating T cellsspecific for particular antigens of interest and modulating T cellfunction ex vivo which methods use, in various embodiments, flowcytometry and immunoaffinity chromatography. Additionally, the presentinvention is directed to artificial antigen presenting cells (artificialAPCs) and methods of making artificial APCs. In a preferred embodiment,such artificial APCs are used to isolate, expand, and modulateantigen-specific T cells. Additionally, the present invention isdirected to methods of treating conditions which would benefit from themodulation of T cell responses, for example, transplantation therapies,autoimmune disorders, allergies, cancers and viral infections orvirtually any T cell mediated disease. The present invention is furtherdirected to a T cell modulation column device as well as a kit forisolating and modulating antigen-specific T cell populations.

Artificial APCs and APC content

In one aspect, artificial APCs are provided having a syntheticmembrane-based vesicle, such as a liposome containing cholesterol andneutral phopholipids such as phosphotidylcholine, that functions as anAPC having capacities equivalent to a natural APC to bind to and inducean antigen-specific T cell response. Such an artificial APC comprisesmultiples of homo- or heterogenous combinations of MHC: antigencomplexes incorporated therein as well as other functional moleculesincluding accessory molecules, co-stimulation molecules, adhesionmolecules, and other immunomodulatory molecules such as cytokines,cytokine receptors, chemokines, and chemokine receptors. Additionally,these APCs may include a mechanism to properly orient these molecules ofinterest in the APC membrane.

In one embodiment, accessory molecules may be used to facilitate andstabilize the interaction between the antigen specific T cell and theMHC: antigen complex. In this embodiment, an example of an accessorymolecule is LFA-1. Other accessory-molecules include, but are notlimited to CD11a/18, CD54(ICAM-1), CD106(VCAM), and CD49d/29(VLA-4), aswell as antibodies to each of these molecule's ligands.

In another embodiment, the artificial APC includes co-stimulatorymolecules that function to stimulate or activate an antigen-specific Tcell. One form of activation is cell proliferation. Suitableco-stimulatory molecules include, but are not limited to, B7-1, B7-2,CD5, CD9, CD2, CD40 and antibodies to their ligands. Preferably, suchco-stimulatory molecules can be produced by recombinant methods.Co-stimulatory molecules can be used for a variety of purposes inaddition to eliciting cell proliferation. For example, it is known thatmemory CD4+ T cells express B7-2 whereas naïve CD4+ T cells do not.Neither type cell expresses B7-1 (Hakamada-Taguchi, R. European Journalof Immunology. 28:865–873.). Thus, the current invention may be used toselectively target memory T cells by incorporating anti-B7-2 into theartificial APC complex.

In another embodiment, the artificial APC also includes adhesionmolecules to facilitate strong and selective binding between theartificial APC and antigen-specific T cells. Suitable adhesion moleculesinclude, but are not limited to, proteins of the ICAM family, forexample ICAM-1 and ICAM-2, GlyCAM-1, as well as CD34, anti-LFA-1,anti-CD44 and anti-beta7 antibodies, chemokines, and chemokine receptorssuch as CXCR4 and CCR5, and antibodies to Selectins L, E, and P. Suchmolecules are known to be important as homing molecules for cellsdestined for specific locations in vivo. For example, Alpha4beta7 andL-selectin have been proposed as gut and peripheral lymphnode homingmolecules respectively. Alpha4beta7 is expressed mainly on memory Tcells while L-selectin is expressed mainly on naïve T cells (Abitorabi,M. A. J Immunol. 156:3111–3117.). It is also known that endothelialselectins (E-selectin and P-selectin) are associated with theextravasasion of T cells into inflammatory sites in the skin (Tietz, W.J Immunol. 161 :963–970.). In the current invention a beta7 bindingmolecule and gut addressin MAdCAM-1, or an anti-L-selectin antibody maybe incorporated into the artificial APC to distinguish further the typeof T cell binding to the MHC:antigen complex.

In another example, it is known that CD44, which binds hyaluronan, isinvolved in Anti-CD44 antibody will bind to CD44 and strip it from theleukocyte surface. In one embodiment of the invention, anti-CD44 isincorporated into an artificial APC for use in stripping CD44 fromleukocytes as desired thereby helping to inhibit the extravasation ofthe cells into extracellular spaces once the treated cells are returnedto the patient. In another embodiment the anti-CD44 can be infused intoan immunomodulatory column where the leukocytes have been captured byartificial APCs for the same purpose.

In another embodiment, other functional molecules (i.e., modulationmolecules) may be incorporated into the artificial APC to facilitate Tcell modulation. Examples of such molecules which may be incorporatedinto the artificial APC include, but are not limited to, CD72, CD22, andCD58, or antibodies to their ligands, antibodies to cytokine orchemokine receptors or small molecules which mimic the actions of thevarious cytokines or neuropeptides. These modulation molecules may beused for example to modulate the phenotype of antigen-specific T cells.

In another embodiment of the invention, the artificial APCs may alsocomprise irrelevant molecules which are included for the purpose ofproviding a means to anchor the APC to a solid support or to carry alabel. Such molecules are termed irrelevant because they do not interactwith the binding, activation, or modulation of the T cells.

In still another embodiment, any of the aforementioned molecules ofinterest (i.e., MHC, functional, accessory, irrelevant) may be bound toa cholera toxin subunit moiety by either a linking moiety of by arecombinant construction of a fusion peptide wherein the toxin subunitis linked directly to the protein of interest. In such embodiment, thecholera toxin subunit is positioned with respect to the molecule ofinterest such that the active portion of the molecule of interest isavailable for contact with T cells while the toxin portion remains inthe nonpolar region of the lipid layer of the APC. In a preferredembodiment, the cholera toxin moiety remains in the APC's interior bybinding to GM-1 that is incorporated into the APC's lipid interior.

In still another embodiment, the APC comprises phospholipids,cholesterol, and GM-1 molecules each present in an appropriate ratio toallow free migration of molecules of interest around the lipid layer.Phospholipids contemplated include neutrally charged phospholipids suchas phosphotidylcholine and cholesterol. Additionally, the cholesterolprovides a surfactant property allowing the phospholipid to carry thevarious molecules of interest (accessory, irrelevant, modulation,adhesion, and co-stimulatory) in a manner that aids the free mobility ofsuch molecules within the liposome membrane layer without disruption ofthe membrane in ex vivo environments.

In still another embodiment, the APC comprises antigens wherein theantigens are presented by an MHC components for contact with andrecognition by a T cell receptor. Such antigens may be selected from thegroup consisting of a peptide, a peptide derived from the recipient forgraft versus host diseases, a cancer cell-derived peptide, a peptidederived from an allergen, a donor-derived peptide, a pathogen-derivedmolecule, a peptide derived by epitope mapping, a self-derived molecule,a self-derived molecule that has sequence identity with saidpathogen-derived antigen, said sequence identity having a range selectedfrom the group consisting of between 5 and 100%, 15 and 300%, 35 and100%, and 50 and 100%.

In still another embodiment, the APC comprises labels wherein a label isassociated with at least one of the group selected from the groupconsisting of a lipid bilayer of the liposome components, a lipid of theliposome, an antigen, an MHC molecule, a co-stimulatory molecule, anadhesion molecule, a cell modulation molecule, GM-1, cholera toxin βsubunit, an irrelevant molecule, and an accessory molecule.

Artificial APC Formation

In a preferred embodiment, artificial APCs may be made by:

-   -   (a) obtaining MHC:antigen complexes of interest;    -   (b) combining said MHC:antigen complexes and accessory molecules        such as ICAM-1, with an artificial lipid membrane comprising the        aforementioned lipids, cholesterol, and GM-1 molecules to form        membrane-associated MHC:antigen:accessory molecule complexes,        (i.e., liposome:MHC:antigen:accessory molecule complexes); and    -   (c) combining said liposome:MHC:antigen:accessory molecule        complexes resulting from step (b) with one or more types of        functional molecules (i.e.), other accessory molecules,        co-stimulatory molecules, adhesion molecules, modulation        molecules, and irrelevant molecules) to form an artificial APC        comprising liposome:MHC:antigen:accessory molecule:functional        molecule complex. Preferably, steps (b) and (c) are performed        simultaneously. Additionally, in this embodiment example, as        well as all others mentioned herein, each of these molecules        incorporated may be prelinked to cholera toxin (as by fusion        protein construction or linker moiety).

In one embodiment, the functional molecules are individually optional.In another embodiment the irrelevant molecules are optional.

In another preferred embodiment, the artificial APCs may be made by:

-   -   (a) obtaining a spheroid solid support of interest having        affinity for non-polar regions of a phospholipid; and    -   (b) combining MHC:antigen complexes, accessory molecules such as        LFA-1, and functional molecules (i.e., other accessory        molecules, co-stimulatory molecules, modulation molecules,        irrelevant molecules, and adhesion molecules) with the        phospholipid, cholesterol, GM-1 components and solid support to        form a solid support        associated:membrane-bound:MHC:antigen:accessory        molecule:functional molecule complexes (i.e., solid        support:phospholipid:MHC:antigen:accessory molecule:functional        molecule complex) wherein of the molecules of (b), none are        covalently bound to the solid support except optionally the        lipid component.

In this embodiment, the solid support is preferably a glass bead ormagnetic bead. It is also preferred that the phospholipid bephosphotidylcholine. In one embodiment of this aspect, the functionalmolecules are individually optional. In another embodiment, the solidsupport is either a glass or magnetic bead and has a diameter of between25 and 300 μm. Additionally, another solid-support APC construct hasonly lipids, cholesterol and a capture moiety having affinity forcapturing an irrelevant molecule that is located on a non-solid-supportAPC. In such construct, the lipid layer is generally a monolayer.

Artificial APC Methods of Use

In another embodiment, the present invention is directed to a method ofisolating T cells specific for an antigen of interest using anartificial APC comprising:

-   -   (a) obtaining a biological sample containing T cells which are        specific for an antigen of interest;    -   (b) preparing a liposome:MHC:antigen:accessory        molecule:functional molecule complex (i.e. artificial APC),        wherein the antigen in said complex is said antigen of interest;    -   (c) contacting the biological sample obtained in step (a) with        the artificial APC obtained in step (b) to form a artificial        APC:T cell complex;    -   (d) removing said complex formed in step (c) from said        biological sample; and    -   (e) separating T cells specific for said antigen of interest        from said complex formed in step (c).

Optionally, such a method of isolating T cells specific for a particularantigen of interest may include the step of determining the quantity ofsuch T cells complexed with the artificial APC, and/or may include thestep of characterizing the functional phenotype of such T cells.Preferred biological samples containing T cells specific for an antigenof interest include bodily fluids such as blood, blood plasma, andcerebrospinal fluid. Other suitable biological samples include solidtissue, for example histological specimens.

In a preferred embodiment, the method uses FACS technology, and theantigen is labeled. Preferred labels include biotin, fluorochromes andradioactive labels. For example, one type of label which may be used isvancomycin (Rao et al., Science, Vol. 280:5364:708–11,1998). In anotherembodiment of the method, also using FACS technology, the liposome maybe labeled or the label may be noncovalently enclosed within theliposome matrix. If the label is within the liposome matrix, the labelmay be either enclosed within the liposome or incorporated within thelipids of the outer membrane of the liposome. In another embodiment, theirrelevant molecule, if present, may be labeled. In yet anotherembodiment, the complex of the artificial APC and T cell may be removedfrom the biological sample by capturing the complex via the irrelevantmolecule on to a solid support. In such case, a solid support comprisesan irrelevant molecule binding or capture molecule (e.g. anti-irrelevantmolecule antibody) bound either directly to the solid support ornoncovalently associated with a phospholipid bound to the support.

In another embodiment, the present invention provides an alternatemethod of isolating T cells specific for an antigen of interest. Thisalternate method comprises:

-   -   (a) contacting an artificial APC having a MHC:antigen:accessory        molecule component of interest with a solid support to form a        solid support artificial APC (The liposome of the APC contains a        binding molecule i.e., an “irrelevant” binding molecule. The        capture molecule that captures the irrelevant molecule may be        bound to said solid support via a linker or may be associate        with a phospholipid layer on the solid support). In this        embodiment, the antigen binding region of said MHC:antigen        component is available for binding to a T cell receptor without        steric hindrance because the MHC:antigen component is free to        move within the liposome membrane of the APC while the        irrelevant binding protein allows the APC to be anchored to the        solid support;    -   (b) contacting said solid support artificial APC with a        biological sample containing T cells specific for an antigen of        interest to form a solid support artificial APC:T cell complex;    -   (c) removing said solid support: artificial APC:T cell complex        from said biological sample; and    -   (d) separating the T cells specific for said antigen of interest        from said complex.

In a further aspect of the present invention, kits for the isolation ofT cells specific for an antigen of interest are provided. In oneembodiment, the kits comprise:

-   -   (a) APCs and solid supports such that there is included APCs        having MHC (and other functional molecule where desired)        complexes; and    -   (b) materials well known to those knowledgeable in the art which        facilitate the completion of the isolation of an        antigen-specific T cell population including, but not limited        to, buffers, culture medium, (included in buffers may be        cytokines, antibodies to various transmembrane or soluble        molecules, chemokines, neuropeptides, or: steroids), (included        in culture medium may be cytokines, antibodies to various        transmembrane or soluble molecules, chemokines, neuropeptides,        or steroids), antigens, MHC molecules, accessory molecules,        co-stimulatory molecules, modulatory molecules and adhesion        molecules.

In another kit embodiment, the kit may comprise a solid support having ameans to capture an irrelevant molecule located in an artificial APC,and an artificial APC constructed as described above. In anotherembodiment, the kit may comprise virtual artificial APCs or solidsupports comprising a lipid layer.

In another preferred embodiment, the invention includes anantigen-specific T cell isolation and modulation column device. In thisembodiment, the device comprises compartments that may be isolated fromone another having entrance and exit flow ports between saidcompartments and between the compartments and external apparatuses. Anyof the compartments of such column device may further comprise solidsupports capable of binding irrelevant molecules of artificial APCs orsolid supports that function directly as artificial APCs as describedabove. Moreover, such a device may be used in connection with solubleimmunomodulatory molecules that are neither bound to a solid support orincorporated into an artificial APC. Examples of such molecules includecytokines, chemokines and hormones.

In another such example, if leukopheresis is being performed with theintention of reintroducing the cells back into the patient's body,soluble factors may be introduced into a column device to induceproduction of IL2 in naïve T cells, IL2 being necessary for T cellgrowth. Likewise, IL4 or soluble IL4 receptor antibody may be introducedinto an immunomodulatory column to enhance the Th2 phenotype in specificT cells of interest.

The invention further comprises a method of modulating T cell responses(i.e., altering a T cell's phenotype). In such embodiment, methods ofregulating or modifying T cell responses ex vivo, such as in a columndevice of the invention, are provided comprising the steps of isolatingT cells which are specific for an antigen of interest and combining saidisolated T cells with an artificial antigen presenting cell. Theaforementioned steps may be performed simultaneously or separately as byaddressing antigen-specific T cells from one compartment to anotherafter first capturing the T cell followed by introduction of anartificial APC. Preferably, the T cells specific for an antigen ofinterest are isolated using the T cell isolation methods describedabove.

The modulation of T cell response may comprise changing, in whole or inpart, the functional pattern of cytokine receptor expression, cytokineproduction, chemokine production, and/or chemokine receptor expressionby the isolated T cells specific for a given antigen. For example, a Tcell may be stimulated to shift its phenotype from a Th0 to a Th1. Inanother example, a T cell may be stimulated to shift its phenotype froma Th1 response to a Th2 response. In yet another example, a T cell maybe stimulated to shift from any other phenotype to a Th3 phenotype.Amongst the many possible means to induce modulation of a T cellresponse for the purpose of increasing its Th2 response and/or decreaseits Th1 response, preferably the artificial APC used in such methodexpresses the co-stimulatory molecule B7-2.

In another embodiment, the modulation of T cell response may comprisechanging, in whole or in part, the functional pattern of cytokineproduction by said isolated T cells from a Th2 response to a Th1response. Preferably, to modify a T cell response to induce it toincrease its Th1 response and/or decrease its Th2 response, theartificial APC used in such method expresses the, co-stimulatorymolecule B7-1.

In another example of T cell modulation, it is known that ST2Lexpression is Th2-type specific. In the current invention, ST2L may beincluded in the APC in order to identify, isolate and extractantigen-specific T cells of the Th2 phenotype and/or enrich T cellpopulation for antigen-specific Th1 phenotype in the treatment ofautoimmune disease.

In another example, OX40 ligand is known to induce a Th2-like phenotypein naïve T cells (Flynn, S. Journal of Experimental Medicine.188:2:297–304.). In the current invention, OX40 may be incorporated intoand artificial APC to selectively induce a Th2 phenotype.

In another example, CD30 is known to have association with asthma(Spinozzi, F. Mol Med. 1:7:821–826.). In the current invention, CD30 maybe incorporated into artificial APCs to identify, isolate and removeantigen-specific T cells of the Th2 phenotype or to augment T cellresponse away from harmful T helper cells in the treatment of allergicconditions.

In yet another embodiment, the modulation of T cell response maycomprise inducing anergy and/or apoptosis. Since it is known that thesame cell need not present both the specific antigen and theco-stimulatory molecule for T cell activation (Ding, L and Shevach, E.M. European Journal of Immunology. 24:4:859–866.), our system isapplicable to situations where the artificial APC does not express aco-stimulatory molecule, or contains another effector molecule, e.g. Fasligand, to induce anergy. Thus, the artificial APC used in such methodwould not express a co-stimulatory molecule but may alternativelyexpress Fas ligand.

In yet another embodiment, the modulation of T cell response maycomprise inducing T cell proliferation in general, without regard tomodifying Th1 or Th2 response, and without regard for inducing anergy.Preferably, this is accomplished by an artificial APC that expresses ananti-CD28 antibody.

In yet another preferred embodiment, the present invention providesmethods of treating a condition in a subject who would be benefited bymodulating the functional pattern of active factors expressed by a Tcell. Such method of treatment could include in addition to the use ofartificial APCs, the use of a column device described herein. Forexample, in such a treatment regimen, production of cytokines by a Tcell may be modified in certain antigen-specific T cells to increase Th2response and/or decrease Th1 response. In such a method, a subject's Tcells that are specific for an antigen capable of triggering a Th1response are isolated by contacting said cells with an APC having anMHC:antigen complex containing an appropriate antigen. By also includingthe co-stimulatory molecule B7-2 on the APC, the T cells may be directedto modify their response and cytokine production to increase a Th2response. Conditions which would be benefited by altering the functionalpattern of response toward a Th2 response include, for example,autoimmune diseases such as type 1 diabetes mellitus, multiplesclerosis, rheumatoid arthritis, dermatomyositis, juvenile rheumatoidarthritis and uveitis.

In another example of a method of treatment, it is known that thecross-linking of the CD40 ligand by means of antibodies induces cellproliferation and IL4 production (Blotta, M. H. J. Immunol.156:3133–3140.). Additionally, it is known that blockade of CD40/CD40ligand pathway induces tolerance in murine contact hypersensitivity(Tang, A. European Journal of Immunology. 27:3143–3150.). In the currentinvention, CD40 or anti-CD40 ligand antibody may be incorporated into anartificial APC to induce T cell modulation toward production of IL4and/or tolerance to alleviate inflammatory autoimmune disorders.

In yet another example of a method of treatment, a subject may bebenefited by altering the functional pattern of cytokine production bycertain antigen-specific T cells to increase Th1 response and/ordecrease Th2 response. Such methods comprise isolating a subject's Tcells that are specific for an antigen capable of triggering a Th2response by contacting said cells with an APC containing an MHC:antigencomplex having an appropriate antigen, wherein said artificial APC alsoexpresses the co-stimulatory molecule B7-1. Conditions which would bebenefited by altering the functional pattern of cytokine production toincrease Th1 response and/or decrease Th2 response include, for example,allergy, for example allergy to dust, animal skin bypass products,vegetables, fruits, pollen and chemicals. Other conditions which may bebenefited include cancers and some types of infections (e.g., viral,protozoan, fungal and bacterial).

In another example of a method of treatment, it is known that anti-B7-1antibody will reduce the incidence of EAE, an animal model of multiplesclerosis. Anti-B7-2 antibody is known to increase the severity of EAE,while co-treatment with anti-IL4 antibody will prevent diseaseamelioration. In the current invention, more than one level of controlwith respect to this disease is possible. For example, artificial APCsmay be generated that express anti-B7-1 antibody and/or IL4 to elicit Tcell response favorable to treating the disease.

In another example, a regimen may be developed for treating melanoma byscreening for T cell responses to epitopes derived from MAGE-1, MAGE-3,MART-1/melan-A, gp100, tyrosinase, gp75, gp15, CDK4 and beta-catenin,all of which are known to be associated with the disease. T cells havingspecificity for these molecules may be activated and modulated invarious ways. For example, T cells that are specific for a unique cancerrelated antigen can act to cause destruction of the cancerous cells maybe proliferated and infused into a patient.

In another example of a method of treatment, artificial APCs may bedesigned to augment antigen-specific T cell response away from theharmful helper type T cells or used to deplete offending T cells in thetreatment of multiple sclerosis. Such depletion or modulation of the Tcells may be carried out in combination with the infusion into a columndevice of either Fas, Fas Ligand, anti-Fas or anti-Fas ligand antibody.Additionally, artificial APCs may be designed to incorporate SLAMreactive molecules into the liposome complex which functions to induce asuppressive phenotype.

In another treatment example, SLAM reactive molecules incorporated in anartificial APC may be used to treat Th2-mediated autoimmune diseases bymodulating the T cell response to shift from a Th2 profile to a Th0/Th1profile while at the same time inducing IL-2 independent, co-stimulationindependent proliferation.

In yet another treatment example, cartilage degradation that isassociated with rheumatoid arthritis can be prevented by use of IL-10and IL-4 in an artificial APC or by infusion of the soluble moleculesinto a column device to modulate antigen-specific T cells away fromactivated Th1 state.

In yet another treatment example, IL-12 can be used such as by infusioninto a column device or incorporation in an artificial APC to induce Th1type inflammatory response to help treat Th2 mediated autoimmunediseases.

In another example of a treatment method, T cell mediated milkintolerance may be treated by isolation and depletion of T cellsspecific for the allergen which is incorporated as an MHC bound antigenon an artificial APC.

In still another example of using the invention to treat cancerousconditions, T cells specific for the ras peptide, or variants thereof,may be stimulated by response to an artificial APC containing the raspeptide in the treatment of cancerous cells expressing the ras mutation.

In another example of the advance of the current invention over that ofcurrently applied art, instead of observing the effects of specificmolecules on T cell response in vivo, the current invention allows oneto follow T cell modulation ex vivo. For example, previous studies(Gaur, A. J of Neuroimmunol. 74:149–158.) showed that using I.V.injection of non-encephalitogenic APL91 peptide of MBP amelioratesdisease by shifting cytokines from Th1 to Th2 phenotype. The same typeof injections using encephalitogenic superagonist APL A97 amelioratesdisease by causing deletion of specific T cells. The problem arises thatunderstanding the bioavailability of the injected complexes is difficultat best while the ex vivo methodology of the current invention allowsone to follow the specific actions of the peptides when used either inan artificial APC or in soluble form in a column device.

In another situation, prior studies have indicated that in vivoapplication of peptides to treat autoimmune disease states is related toepitope spreading resulting in relapsing episodes of disease (Lehmann,P. V. Nature. 358:6382:155–157.), (McRae, B. L. Journal of ExperimentalMedicine. 182:75–85.). The ex vivo application of the current inventionis preferred because specific peptides can be used to isolate andidentify antigen-specific T cells without exposing a patient to thedanger of epitope spreading that is associated with relapses of certainautoimmune diseases.

In a further preferred embodiment of the invention, a method ofidentifying T cells that express MHC epitopes important to graft versushost rejection in transplantation therapy is provided. In thisembodiment, such MHCs are identified followed by their incorporationinto the artificial APC. Such APCs may be used to capture and depletethe recipient's T cells having specificity for such epitopes so as toallow a favorable modulation of the recipient's immune response to thegraft. More specifically, peptides derived from the MHC of a recipientare bound to the MHC of a donor that are incorporated into artificialAPCs. Such APCs are used in combination with tolerogenic stimuli also onthe APC or infused into the immunomodulatory column. The donor's T cellsare then screened to bind reactive T cells that can then be discarded.In a preferred embodiment, the column device of the invention can beused to carry out such immunoleukophoresis.

In another embodiment, the method contemplates treatment of anindividual to cause a favorable immune modulation to an allograftcomprising:

-   -   (a) predicting a donor's MHC to which a recipient may react;    -   (b) testing the predicted MHC epitopes with a recipient's T        cells to identify antigenic epitopes;    -   (c) using identified epitopes in an artificial APC to deplete        the recipient's antigen-specific (i.e., donor-specific) T cells        while additionally desensitizing the recipient to the epitope by        contacting the recipient (as by feeding or nasal injection) with        increasing doses of the donor-specific antigen.

In still another preferred embodiment, the invention provides a methodof identifying an individuals' MHC epitopes that are of import inimmunologic responses to pathogenic agents. In this embodiment, anindividual's MHC is screened for epitopes that have appreciable sequenceor molecular structure recognition with pathogen-derived molecules. Theidentified MHC epitopes can be used to elicit immunity that may beemployed directly as a vaccine against such MHC epitopes that havesequence recognition with pathogen-derived peptides. For example, such avaccine may be used to reduce natural APCs that express MHC moleculesassociated with autoimmune diseases including, but not limited to,multiple sclerosis, rheumatoid arthritis, and diabetes. In anotherpreferred embodiment, the identified MHC epitopes can be incorporatedinto a liposome structure as a co-stimulatory molecule to enhance theeffect of artificial APCs that express other disease related antigens.

In yet another preferred embodiment, antigenic moieties of the pathogenwhich have or are likely to have MHC mimics may be used in artificialAPC MHC:antigen complexes to isolate T cells specific for suchpathogen-derived antigenic motifs or mimics thereof for the productionof a T cell vaccine. Such a vaccine may be used to directly fightprogression of an infection or disease caused by a pathogen or that isthe result of a pathogen-derived antigen induced autoimmune associatedinflammatory response. Usually a self-derived molecule that mimics apathogen-derived antigen comprises a polypeptide. As used herein such amimic has an amino acid sequence identity with said pathogen-derivedantigen to an extent necessary for the MHC to bind to the mimic. Therange of sequence identity may be anywhere between 5 and 100% dependingupon which amino acids in a peptide sequence elicits either recognitionby the MHC and/or stimulation of a T cell response. Generally, the rangeis between 5 and 100%, usually, the range is between of a range of 15and 100%, preferably the range is between 35 and 100%, and mostpreferably the range is between 50 and 100%.

In still other embodiments, the invention provides a means to addressother immunologic conditions relevant to T cell response. For example,apoptosis of T cells induced by MHC molecules through the CD95/CD95ligand pathway can be controlled by incorporating anti CD95 into anartificial APC and modulating antigen-specific T cells.

In another application, dendritic cells important to immune response maybe manipulated ex vivo in the same fashion as T cells in the manyexamples provided above.

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawing(s) will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee. The invention will be further described with reference to theaccompanying drawings in which:

FIG. 1 is a schematic representation of the basic features of theinvention and it's interaction with various molecules on a T cell, where(1) is an artificial APC who's embedded molecules have conformed to thecapping of the T cell, where (2) is an artificial APC that has notinteracted with a T cell and who's molecules are randomly dispersedthrough the membrane, where (T) is a T cell who's molecules have cappedin response to the interaction with the artificial APC, where (3) is anaccessory molecule for stabilizing the binding between the TCR andMHC:antigen complex, where (4) may be T cell accessory or adhesionmolecule ligands, where (5) may be T cell co-stimulatory moleculeligands, where (6) may be an MHC:ag complex, where (7) may be cytokinerelated molecules, where (8) may be chemokine related molecules, where(9) may be an irrelevant molecule used in binding of the artificial APCto a solid support or a molecule which is tagged with a molecule used invisualization.

FIG. 2 is a schematic representation of an embodiment of the inventionrepresentative of a portion of an artificial APC (10) in which thepeptide (11) complexed to the MHC (12) is tagged, wherein the tag, forexample, is biotin (13). The visualization of the complex comprising theabove components may occur (while a TCR (14) on a T cell (15) is boundto the APC) by FACS through the addition of a streptavidin moleculecomplexed to a fluorochrome (16).

FIG. 3 is a schematic representation of an embodiment of the inventionrepresented by a portion of an artificial APC (17) interacting with a Tcell (18) through the TCR (19), using the artificial APC's MHC (20) andunlabeled peptide (21). The visualization by FACS is performed by theinclusion of an irrelevant molecule (22) having an attached label (23).The irrelevant molecule minus the label may also be used to bind theliposome to a solid support.

FIG. 4 is a schematic representation of an embodiment of the inventionrepresented by a portion of an artificial APC (24) interacting with a Tcell (25) through the TCR (26), using the artificial APCs MHC (27) andunlabeled peptide (28). A fluorochrome (29) may be added to the dialysisbuffer during the formation of the liposomes so that visualization maybe carried out by FACS.

FIG. 5 is a schematic representation of an embodiment wherein theartificial APC (30) includes a functional molecule (31) (such as anaccessory, co-stimulatory, adhesion, modulation, cytokine, or chemokinemolecule) that interacts with a molecule (33) expressed by a T cell 32.

FIG. 6 is a schematic showing the capture of artificial APCs (35) and(37) by capture molecules (39) which are noncovalently associated with alipid layer (38) (e.g., a neutral phospholipid) attached to a solidsupport (34).

FIG. 7 is a schematic showing other embodiments of artificial APCdesigns wherein FIG. 7 a represents a solid support (40) having anon-covalently associated lipid layer (42) containing variouscomponents; of artificial APCs (41) that bind a T-cell (43). FIG. 7 bshows a solid support (44) having the various components of artificialAPCs (45) without a lipid component that can bind to a T cell (46).

FIG. 8 shows a column device having a multiplicity of compartments forprocessing antigen-specific T cells. The column, as shown, also containsAPCs and various embodiments of solid supports.

FIGS. 9A and B is a series of FACS figures detailing the specificity ofthe embodiment of the method diagramed in FIG. 2 using two differentmurine hybridomas specific for the OVA peptide in the context of eitherI-A^(d) or I-A^(S). The Hi 15 peptide has two identities and oneconservative substitution with the OVA peptide, thus being a stringentcontrol of specificity.

FIG. 10A, B, and C shows a series of experiments from a non-transgenicmurine model in which the characterization of the antigen specificity(the Iα52 peptide) of a T cell population without the use of the currentinvention was attempted.

FIG. 11 shows the ability of the invention to determine directly theantigen specificity of a T cell population in the non-transgenic mousemodel that could only be inferred in FIG. 10.

FIG. 12 shows the ability of the invention to determine thecross-reactivity of a singular group of antigen-specifically defined Tcells seen first in FIG. 10, then in FIG. 11. Such a techniquerepresents an improvement over prior art, as internalized antigen cannotbe removed.

FIGS. 13A–E is a series of FACS figures showing the use of the inventionto identify T cells based on the specificity of the TCR in the PBMC of apatient with RA.

FIGS. 14A–D is a series of FACS figures showing the use of the inventionto identify T cells based on the specificity of the TCR in the PBMC of apatient with JDM.

FIG. 15 shows the identification of antigen specific cells with T cellcapture in a monoclonal TCR population wherein OVA³²³ ^(—) ³³⁹/IA^(d)specific T cell hybridoma 8DO cells were incubated with artificial APCscomplexed with IA^(d) and biotinylated OVA³²³ ^(—) ³³⁹. Prior to theincubation, the hybridoma cells were stained with CD4 PE (phycoerithrin,i.e., a fluorochrome) and the artificial APCs, complexed with IA^(d) andbiotinylated OVA³²³ ^(—) ³³⁹, were stained with streptavidin labeledwith CY. As control for specificity of the binding, T cells of the samehybridoma were incubated with liposomes complexed with IE^(d) andbiotinylated OVA³²³ ^(—) ³³⁹. Binding of hybridoma T cells to artificialAPCs could be inhibited by co-incubation with monoclonal anti-IA^(d)antibody. The histograms show the intensity for staining forstreptavidin CY in CD4 gated hybridoma cells. Gates were set onirrelevant isotype controls for CD4, and on binding ofcychrome-conjugated streptavidin to T cells/artificial APC withunbiotinylated OVA³²³ ^(—) ³³⁹ peptide.

FIGS. 16(A–D), 17(A–D), and 18(A–D) are color photos showing that theinteraction of T cells with artificial APCs allows physiologicalmigration of TCR proteins toward the interaction side, i.e., “capping”.Resting 8DO T cells were stained with FITC-cholera toxin, incubated withartificial APCs presenting IA^(d)/OVA³²³ ^(—) ³³⁹ complexes and analyzedby confocal microscopy. Panels A and B of each of FIGS. 16–18 show thered and green fluorescent dyes, respectively. Panel C shows the cells asseen by phase contrast. Panel D is the combination of the twofluorescent dyes with phase contrast. Panel D shows the combinedfluorescence of the red and green dyes so that co-localization (FIG.16D), and capping (FIGS. 17D, and 18D) are observed. Specifically, FIGS.16A–D shows 8DO cells alone stained with Alexa 568-anti CD3 (red) boundto the T cell receptor and FITC-cholera toxin (green) bound to thecholera toxin. Thus, FIGS. 16A–D shows that the T cell receptor isassociated with the cholera toxin antigen. This further shows thatcholera toxin is a good tool for identifying the T cell receptor in thissystem. FIGS. 17A–D shows 8DO cells incubated for 20 minutes withartificial APCs expressing IA^(d)/OVA³²³ ^(—) ³³⁹ complexes. ArtificialAPCs were stained with Alexa568 anti MHC (red) (i.e., the MHC is labeledred), and FITC cholera toxin (green) (i.e., the cholera toxin bound tothe T cell receptor is labeled green). Thus, FIGS. 17A–D shows that theinteraction between T cells and artificial APCs allows physiologicmigration and capping of the T cell receptor in the T cell membrane.FIGS. 18A–D shows 8DO cells incubated for 20 minutes with artificialAPCs expressing IA^(d)/OVA³²³ ^(—) ³³⁹ complexes. Artificial APCs lipidmembranes were labeled using FITC (green). The T cell receptor werelabeled with Alexa568 anti CDS (red). Thus, FIGS. 18A–D further confirmsthe data shown in FIGS. 17A–D.

FIGS. 19A–D are PAGE photos showing optimization of peptide (antigen)loading of human and mouse MHC class II molecules wherein detergentsolubilized MHC class II molecules were incubated with biotinylatedpeptides at the designated pH and temperature for 16 and 24 hrs. Peptideloading was analyzed through ECL. FIGS. 19A and B show binding ofbiotinylated PADRE peptide to HLA-DR4. FIGS. 19D and C show binding ofbiotinylated OVA 323–339 to mouse IAd. The photos show output signal ofthe level of binding of the peptides to MHC. Results indicate that anincubation of 16 hrs at room temperature and pH 7 and a molar ratio of200 to 1 provide for optimal results with respect to the human MHC and1000 to 1 for the mouse.

FIG. 20 is a graph showing characterization of the artificial APCs byflow cytometric analysis wherein the approximate size of thefluorescent-labeled liposomes was determined through comparison withsingle size fluorescent particles with a mean size between 0.05 μm and0.85 μm. The 0.85 um labeled curve represents beads of 0.85 μm diameter,while the curve labeled 0.05 represents beads of 0.05 μm in diameter,the shaded curve represents the size of APC.

FIG. 21 is a graph showing determination of the optimum incorporation ofMHC class II in liposomes. Optimum incorporation is a factor of theratio of the phospholipid and cholesterol. The ratio of the lipidcomponents were tested such that fluorescent labeled liposomes werecomplexed with HLA DR4 where the ratio was from 3.5:1 (w/w) to 14:1 andstained with anti-HLA DR PE. The incorporation of DR4 was measured bymeans of flow cytometric analysis of the signal for HLA DR-PE in FL2(X-axis). As shown the number of incorporating events is highest (xaxis) at a lipid/cholesterol ratio of 7:1.

FIG. 22 is a graph showing incorporation of biotinylated PADRE influorescent labeled liposomes, complexed with HLA DR4 and specificity ofthe binding of biotinylated PADRE peptide to HLA DR4 complexed withliposomes. Biotinylated PADRE peptide is incubated with HLA DR4 or MHCClass I, both at a molar ratio of 10:1 (peptide to MHC) prior toincorporation in artificial APCs with fluorescent lipids. As anadditional control, non-biotinylated PADRE peptide and biotinylatedPADRE peptide (at a 1:1 w/w ratio) were incubated with HLA DR4 beforeincorporation in artificial APCs. The PADRE is a pan DR binding peptide.The graph indicates specificity and that label bound to the PADRE doesnot interfere with binding to the MHC component. Specifically wherenon-biotinylated peptide is used at the same time, it will compete outbiotinylated. The Class I binding shows that there must be specificityfor such binding as use of the Class I fails to bind PADRE.

FIGS. 23A–F show plots of FACS analysis showing that T Cell capture isan effective method to identify class II restricted human polyclonal TCells. The panels show a comparison of CD3+ cells binding PADRE/HLA orHA/HLA (HA is hemoglutinin A) complexes. PADRE is used as positivecontrol because it will bind many MHC molecular species therebyproviding an easy means to visualize populations of specific cells. HAis also a pan DR binding peptide but because cells in this example areexpanded using PADRE peptide, use of HA for binding should serve as anegative control. The Y axis represents CD3+ cells; The x axisrepresents HLA/PADRE or HLA/HA complexes. Panels A and B show the % ofPADRE antigen specific T cells at day 0 of culture (A) (i.e., 2.9%), andthe % of antigen specific T cells at day 10 of culture with PADRE (B)(i.e., 8.1%). Thus, cells appear to be expanding as an antigen specificfashion. To test the specificity of the cell population, the % of HAcells are tested. As shown in panels C and D respectively, the % of HAspecific T cells after 0 days of culture with PADRE/HA(C)(i.e. 1.0%)drops to 0.3% HA specific T cells after 10 days of culture with PADRE(D). Panels E and F show the % of antigen specific T cells at day 10 ofculture wherein T Cell Capture using APCs was inhibited with anequimolar ratio of non-biotynilated PADRE. Panel E shows that 50% ofinhibition was achieved, (i.e., the label does not interfere withtesting of specificity using the APC). Panel F shows that 50% ofinhibition was also achieved showing that the T cell/APC binding dependson the specific interaction of the T cell receptor, i.e., inhibition ofbinding using Anti HLA DR antibody at a molar ratio of 0.5:1 antibody toHLA. The FACS plots show results from an experiment wherein PBMC from aHLA DR4 0401+donor were stimulated with 10 μg/ml of PADRE peptide,K(X)VAAWTLKAA (Seq. Id. No. 7) where X is a derivatized amino acid suchas cyclohexylalanine. At days 4 and 7, 10 ng/ml IL-2 was added. At day10 cells were harvested for T Cell capture. HLADR4 was complexed withNBD-labeled liposomes (1:7 ratio HLA to liposomes) through 48 hoursdialysis against PBS in 10.000 M cutoff dialysis membrane (Pierce).Complexes were then incubated for 48 hours at RT with n-terminusbiotinylated peptides at a molar ratio of 10:1 for peptide/HLA. Excessof unbound peptide was removed through 24 hours of dialysis against PBS.Liposome-HLA-peptide complexes were incubated for 30 minutes withstreptavidin-Cy before adding to the cells. The liposome-HLA-peptidecomplexes were then incubated with the stained cells and run on a BectonDickinson FACS Star. Gates were set on viable cells, isotype controlsand cells incubated with streptavidin CY alone.

FIG. 24 is a bar graph showing cytokine production by PADRE-stimulatedcells. Production of IL2 and IFN in vitro of PBMC from HLA DR 0401healthy adults. PBMCs were initially stimulated with 10 μg/ml of Pan DREpitope binding peptide (PADRE) and cultured for 10 days. The cells wererestimulated with autologous APCs and 10 μg/ml of peptide. Culturesupernatants were collected at different days and measured for cytokineproduction by capture ELISA method. The result indicates that cellspecificity can be shown by measuring cytokine production as cellsproliferate. However, measuring cytokine production is actually lessspecific than the method of the invention as demonstrated in FIG. 23.

FIGS. 25A and B are FACS plots showing identification by T cell captureusing artificial APCs of class II-restricted antigen specific mouse Tcells upon immunization and shows increase of IA^(d)/Iα52 specific cellsmeasured after immunization of the mice (at the base of the tail) withIα52 or IFA (adjuvant) alone. Cells were harvested three days after thelast immunization from inguinal draining lymphnodes, stained withanti-mouse CD4 PE and incubated with fluorescein labeled artificial APCscomplexed with IA^(d)/Iα52. FIG. 25A shows IFA only-immunized mice; FIG.25B shows IA^(d)/Iα52-specific CD4 cells. Y axis: CD4; X axis:IA^(d)/Iα₅₂ specific T cells. The result indicates that the adjuvantimmunized cells showed only 0.7% specific cells whereas the Iα52immunized cells comprised 5.4%. Thus, T cell capture using artificialAPCs is useful to show antigen specificity.

FIG. 26 is a bar graph showing T cell proliferation to Iα52 afterimmunization. Cells were harvested from inguinal lymphnodes andincubated for three days with 10 mg/ml of Iα52 peptide. Proliferationwas measured by thymidine incorporation and is expressed as stimulationindex “SI”: cpm of stimulated/unstimulated cultures. The resultindicates that conducting T cell proliferation tests to determinespecificity is effective in measuring the specificity of T cellpopulations. However, the specificity is not to the same extent as thatusing the current invention shown in FIG. 25.

FIGS. 27A–C is a schematic showing methodology for orienting moleculesof interest in the APC liposome matrix. In 27A, a molecule of interestsuch as MHC, functional, accessory, adhesion, or irrelevant molecule,may be synthesized by recombinant methods well known to those skilled inthe art and linked to GM-1 by a linker and properly oriented in the APCmembrane. In 27B, a molecule of interest may be constructed as a fusionprotein with cholera toxin P subunit and the fusion protein anchored inproper orientation in the APC membrane by the cholera toxin moietybinding to GM-1. In 27C, a cholera toxin subunit may be chemicallylinked to SPDP linker (Pierce) and then attached to a molecule ofinterest followed by anchoring to a GM-1 containing APC. In any of theabove, the cholera toxin, GM-1, linkers may be synthetically produced.In the figure, A represents a gene for a molecule of interest, Brepresents the gene for the β subunit of cholera toxin, A1 is anexpression vector, A2 represents expression and isolation of the clonedgene, A3 is an expressed molecule of interest, B1 is a fusion protein ofa molecule of interest and cholera toxin, A4 is a linker, A5 is anartificial APC containing GM-1, A7 is a partial view of an artificialAPC wherein the molecule of interest is directly linked to the GM-1, Cis choler toxin subunit, C1 is a linker, C2 is a molecule of interest,C3 is a choler toxin subunit attached to a linker, C4 is a molecule ofinterest linked to a choler toxin subunit, E represents a liposomebilayer, E1 shows GM-1, E2 is an artificial APC containing GM-1, and Fis a partial view of an artificial APC having a molecule of interestbound to the APC by the binding interaction of the GM-1 and choleramoiety.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to novel methods of isolating T cellsspecific for particular antigens of interest and modulating T cellfunction ex vivo which methods use, in various embodiments, flowcytometry and immunoaffinity chromatography. Additionally, the presentinvention is directed to artificial APCs and methods of makingartificial APCs. In a preferred embodiment, such artificial APCs areused to isolate, expand, and modulate antigen-specific T cells.Additionally, the present invention is directed to methods of treatingconditions that would benefit from the modulation of T cell responses,for example, transplantation therapies, autoimmune disorders, allergies,cancers and viral infections, and virtually any T cell mediated disease.The present invention is further directed to a T cell modulation columndevice as well as a kit for isolating and modulating antigen-specific Tcell populations.

Artificial APC Composition

According to preferred embodiments of the current invention, anartificial APC may comprise MHC:antigen complexes, accessory moleculesand other functional molecules including, but not limited to,co-stimulatory molecules, adhesion molecules, modulation molecules,irrelevant molecules, GM-1 and subunits of cholera toxin attached to anyof the aforementioned molecules, and labels which are collectivelyincorporated within or are associated with the lipids of a liposome orother membrane-based vesicle such as depicted in FIG. 1. Suchincorporated or associated molecules may also include GPI anchoredproteins.

In a first embodiment, the lipid membrane component comprises neutralphospholipids such as phosphotidylcholine and surfactant elements suchas cholesterol. These materials are provided in a proper ratio thatallows the other molecules of interest to freely migrate in the membranelayer. Other lipid membrane components include GM-1 which is atransmembrane pentasaccharide and associates in part with nonpolarregions of the liposome matrix. This GM-1 can be used in associationwith cholera toxin P subunit to orient molecules of interest in theliposome matrix.

Accessory molecules may be included for the purpose of stabilizing theinteraction between a TCR and an MHC:antigen complex. Suitable accessorymolecules may include, but are not limited to, LFA-1, CD49d/29(VLA-4),CD11a/18, CD54(ICAM-1), and CD106(VCAM) and antibodies to their ligands.In a preferred embodiment, the artificial APC includes ICAM-1 as such anaccessory molecule.

Co-stimulatory molecules may be included for the purpose of stimulatingor activating a TCR. Suitable co-stimulatory molecules may include, butare not limited to, B7-1, B7-2, CD5, CD9, CD2, CD40, and antibodies totheir ligands such as anti-CD28.

Adhesion molecules may be included for the purpose of enhancing thebinding association between the artificial APC and a T cell. Suitableadhesion molecules may include, but are not limited to, members of theICAM family such as ICAM 1, ICAM 2 and GlyCAM-1, as well as CD 34,anti-LFA-1, anti-B7, and chemokines such as CXCR4 and CCR5, andantibodies to selectins L, E, and P.

Modulation molecules may be included for the purpose of modulating thephenotype of a T cell. Suitable modulation molecules may include, butare not limited to, CD72, CD22, and CD58 and antibodies to theirligands, antibodies to cytokine or chemokine receptors or smallmolecules which mimic the actions of the various cytokines orneuropeptides.

Irrelevant molecules may be included for the purpose of either carryinga label or serving as a scaffold for binding to a solid support. Such amolecule can be any peptide or other molecule having characteristicsthat make it suitable for use with a liposome and antigen carrier. Suchmolecule should not interfere with the binding of a T cell to theartificial APC.

With respect to the, incorporation of each of the aforementioned MHC,accessory, co-stimulatory, adhesion, modulation, and irrelevantmolecules in the artificial APC, proper orientation of these molecule'sactive centers may be provided by combining the molecules with the βsubunit of cholera toxin so that the cholera toxin subunit can berecognized and bound by GM-1 which is incorporated into the liposomemembrane matrix. The incorporation of the cholera toxin and orientationmechanism markedly increases the ability of the artificial APC tointeract with T cells and other cells and molecules due to the properorientation of incorporated molecules from about 50% without suchorienting to 90% or more with such orienting.

In another embodiment, the aforementioned molecules of interest may beproduced by recombinant technology as is well known to those skilled inthe art. Use of recombinant technology produced molecules furtherprovides the opportunity to produce such molecules as fusion moleculescomprising the molecule of interest attached to the β subunit of choleratoxin. In another embodiment, the recombinantly produced (or for thatmatter a purified natural molecule) may be linked to cholera toxin by acommercial linker.

In still another embodiment, the APC comprises antigens wherein theantigens are presented by an MHC components for contact with andrecognition by a T cell receptor. Such antigens may be selected from thegroup consisting of a peptide, a peptide derived from the recipient forgraft versus host diseases, a cancer cell-derived peptide, a peptidederived from an allergen, a donor-derived peptide, a pathogen-derivedmolecule, a peptide derived by epitope mapping, a self-derived molecule,a self-derived molecule that has sequence identity with saidpathogen-derived antigen, said sequence identity having a range selectedfrom the group consisting of between 5 and 100%, 15 and 100%, 35 and100%, and 50 and 100%.

Examples of some antigens noted above include the peptideQKRAAYDQYGHAAFE (Seq. Id. No. 10) which is derived from E. coli dnaJp1heat shock protein. A human self-derived peptide is QKRAAVDTYCRHNYG(Seq. Id. No. 11) derived from the HLA. This peptide also has sequenceidentity with the pathogen Sequence Id. No. 10. Peptides derived fromepitope mapping include human peptides from the HA I matrix GILGFVFTL(Seq. Id. No. 12), VKLGEFYNQ (Seq. Id. No. 13) which is a HA Inucleoprotein, and PKYVKQNTLKLAT (Seq. Id. No. 14) derived from the HAII locus.

In still another embodiment, the APC comprises labels wherein a label isassociated with at least one of the group selected from the groupconsisting of a lipid bilayer of the liposome components, a lipid of theliposome, an antigen, an MHC molecule, a co-stimulatory molecule, anadhesion molecule, a cell modulation molecule, GM-1, cholera toxin βsubunit, an irrelevant molecule, and an accessory molecule.

Artificial APC Formation

Such artificial APCs may be made by:

-   -   (a) obtaining an MHC:antigen complex of interest;    -   (b) combining said MHC:antigen complex with an artificial lipid        membrane to form a membrane-associated MHC:antigen complex,        (i.e., a liposome:MHC:antigen complex); and    -   (c) combining said liposome:MHC:antigen complex resulting from        step (b) with any of the following: accessory molecule, a        co-stimulatory molecule, an adhesion molecule, a modulation        molecule, and an irrelevant molecule to form an artificial APC        comprising a liposome:MHC:antigen:functional molecule: complex.        Steps (b) and (c) may be performed simultaneously. In one        embodiment of this method, step (c) is optional in whole or in        part with respect to any of co-stimulatory, adhesion,        modulation, irrelevant molecules or GPI proteins. In another        embodiment, any of the molecules of interest (MHC, accessory,        co-stimulatory, adhesion, modulation, irrelevant molecules) can        be bound to the β subunit of cholera toxin and GM-1 can be        included in the APC lipid matrix to provide a means for proper        orientation of the molecules of interest such that their active        centers are oriented to facilitate interaction with T cells and        other components external to the APC.

By “membrane-associated” is meant the non-covalent attraction betweenthe lipid molecules of a liposome and MHCs, antigens, accessorymolecules, co-stimulatory molecules, adhesion molecules, modulationmolecules, irrelevant molecules, GM-1, and cholera toxin β subunit.

In another preferred embodiment, the artificial APCs may be made by:

-   -   (a) obtaining a spheroid solid support of interest having        affinity for non-polar regions of a phospholipid; and    -   (b) combining MHC:antigen complexes, accessory molecules such as        ICAM-1, and functional molecules (i.e., other accessory        molecules, co-stimulatory molecules, modulation molecules,        irrelevant molecules and adhesion molecules) with the        phospholipid and solid support to form a solid support        associated:membrane-bound:MHC:antigen:accessory        molecule:functional molecule complexes (i.e., solid        support:phospholipid:MHC:antigen:accessory molecule:functional        molecule complex).

In this embodiment, the solid support is preferably a glass bead ormagnetic bead. It is also preferred that the phospholipid bephosphotidylcholine. In one embodiment of this aspect, the functionalmolecules are individually optional. In another preferred embodiment,the molecules may be properly oriented by inclusion of a molecule boundto cholera toxin and GM-1 in the APC membrane matrix. In anotherembodiment the complex may include an irrelevant molecule for carrying alabel. In still other embodiments, the antigen may have a label. Instill other embodiments, a label may be noncovalently associated withthe lipid layer.

In yet more embodiments of this solid-support APC construct, the solidsupport is a glass or magnetic bead having a diameter of about between25 to 300 μm. In still another embodiment, the solid-support APCconstruct has only lipids, cholesterol and a molecule having affinityfor binding to an irrelevant molecule that is located on another APC(either solid-support based or non-solid support bases). This constructallows for such molecule (which is also an “irrelevant” molecule) tofloat freely in the lipid layer for proper migration to aid binding ofAPCs that have bound to T cells.

Artificial APC Uses and Methods

In another aspect, the present invention is directed to a method ofisolating T cells specific for an antigen of interest comprising:

-   -   (a) obtaining a biological sample containing T cells which are        specific for an antigen of interest;    -   (b) preparing an artificial APC as described herein comprising        an MHC:antigen component, wherein the antigen in said component        is said antigen of interest;    -   (c) contacting the biological sample obtained in step (a) with        the artificial APC obtained in step (b) to form an artificial        APC:T cell complex;    -   (d) removing said complex formed in step (c) from said        biological sample; and    -   (e) separating T cells specific for said antigen of interest        from said complex.

Any suitable biological sample that contains T cells specific for theantigen of interest may be used in the method. Suitable biologicalsamples containing T cells specific for an antigen of interest includefluid biological samples, such as whole blood, blood cells, blood plasmaand cerebrospinal fluid, and solid biological samples, such as tissue,for example, histological samples. In one embodiment of the aboveexample, the artificial APC may be complexed to a solid support inaddition to the T cell. The complexing of the APC to the solid supportprovides a means to anchor the APC so that it and any T cell binding toit can be preferentially captured and isolated from extraneous matter.In such case, the solid support may be a glass or magnetic bead that iscoated with a lipid monolayer that is bound to the bead by, for example,a linker. The solid support may additionally have noncovalently boundaccessory molecules associated with the lipid layer such as bindingmolecules that recognize and bind to irrelevant molecules associatedwith the artificial APC. In another embodiment, the binding moleculesmay be covalently bound to the solid support by a linker. Additionally,the lipid layer may further include GM-1 ganglioside molecules forbinding to a molecule of interest that is connected to cholera toxinsubunit for orienting said molecule of interest.

By “T cells specific for an antigen of interest” is meant the T cellsexpressing receptors for a relevant target of an immune response. Asnoted above, the specificity of the T cell receptor (TCR) determineswhich antigens bind to the TCR with sufficient affinity to activate aparticular T cell. Optionally, the above outlined method of isolating Tcells specific for a particular antigen of interest may includedetermining the quantity of such T cells that bind to the artificialAPC, or may include characterizing the functional phenotype of such Tcells. Such method may also include the use of a solid supportcontaining a molecule which is able to recognize and bind to theirrelevant molecule located in the lipid layer of the APC. Such solidsupport may further be confined to an accessible chamber of a columndevice.

By “MHC:antigen component” is meant MHC molecules that have affinity forantigens of interest which antigens are associated with such MHC andtogether form a complex of molecules that are inserted into or areassociated with the lipid membrane. Artificial lipid membranes such asliposomes may be prepared in a fashion similar to methods known in theart, (e.g., Watts, et al., PNAS, Vol. 81:7564–68; Buus, et ah, Cell,Vol. 47; 1071:77 herein incorporated by reference). In a preferredembodiment, the liposome forms a bilayer having eukaryotic cell-likeproperties in that non-lipid molecules (e.g. MHC: antigen complexes,GM-1 bound to cholera-molecule of interest) may freely migrate withinthe matrix of the lipid molecules of the liposome's lipid bilayer. Anexample of a liposome:MHC:antigen combination is provided in FIG. 1 andsuch a complex may also be prepared, for example, as described inExample 1 below. Moreover, the lipid bilayer may also include accessorymolecules such as cholesterol to provide elasticity in the bilayer andGM-1 to provide an anchor for orientation of cholera β subunitcomprising molecules of interest.

In one embodiment, a biological sample derived from a tissue sample ofinterest in the form of a single cell suspension is contacted with anartificial APC followed by incubation of the artificial APC tissuesample mixture with antigen-specific staining compounds (i.e. forexample, fluorochrome conjugated antibodies against T cell surfacemarkers of interest e.g., CD3, CD4, or CD8). The cells may also bestained prior to incubation with the complexes. The resulting artificialAPC:T cell complex may be separated from the cell suspension by, forexample, flow cytometry, capture by a solid support in a column device,or centrifugation of such complexes bound to a solid support such asglass beads or magnetic beads.

In one embodiment of the T cell isolation method, the antigen is labeled(FIG. 2). In another embodiment, the irrelevant molecule may be labeled(FIG. 3). In another embodiment of the method, label is associatedeither covalently or non-covalently with the lipid molecules making upthe liposome (FIG. 4). Preferred labels include biotin, fluorochromessuch as FITC, radioactive labels and vancomycin. Use of such labels iswell understood in the art. In embodiments where the label is associatedwith lipids of the liposome, the label may be either enclosed within theliposome or incorporated within the lipids of the outer membrane of theliposome. Preferably, the label is a fluorochrome, for example FITC.Such labeled liposomes may be made by mixing FITC with lipids duringliposome formation, or may be obtained from commercial sources (e.g.,Polar Lipids).

In another embodiment, the present invention provides an alternatemethod of isolating T cells specific for an antigen of interest. Thisalternate method comprises:

-   -   (a) contacting an artificial APC comprising MHC        antigen:accessory molecule component of interest with a solid        support to form a solid support:artificial APC (The liposome of        the APC contains a binding molecule i.e., an “irrelevant”        binding molecule. The capture molecule that binds to the        irrelevant molecule may be bound to said solid support via a        linker or may be associated with a phospholipid layer on the        solid support). In this embodiment, the antigen binding region        of said MHC:antigen component is available for binding to a T        cell receptor without steric hindrance because the MHC:antigen        component is free to move within the liposome membrane of the        APC while the irrelevant binding protein allows the APC to be        anchored to the solid support;    -   (b) contacting said solid support:artificial APC with a        biological sample containing T cells specific for an antigen of        interest to form a solid support:artificial APC:T cell complex;    -   (c) removing said solid support:artificial APC:T cell complex        from said biological sample; and    -   (d) separating the T cells specific for said antigen of interest        from said complex.

In this embodiment, the T cells are separated by either the physicalremoval of the T cell bound solid support from the biological sample, orseparation may occur by retention of the bound T cells in a solidsupport containing compartment of a column device.

In another embodiment, the present invention is directed to otheralternate methods of isolating T cells specific for an antigen ofinterest. One such method comprises:

-   -   (a) obtaining a solid support of interest having affinity for        non-polar regions of a phospholipid;    -   (b) combining the solid support and MHC:antigen complexes,        accessory molecules such as LFA-1, and functional molecules        (i.e., other accessory molecules, co-stimulatory molecules,        modulation molecules, adhesion molecules, and irrelevant        molecules) with the phospholipid support to form a solid support        associated:membrane-bound:MHC:antigen:accessory        molecule:functional molecule complexes (i.e., solid        support:phospholipid:MHC:antigen:accessory molecule:functional        molecule complex);    -   (c) contacting said solid support:phospholipid:MHC:antigen:        accessory molecule:functional molecule complex formed in (b)        with a biological sample containing T cells specific for an        antigen of interest to form a solid        support:phospholipid:MHC:antigen:accessory molecule:functional        molecule:T cell complex;    -   (d) removing said complex formed in (c) from said biological        sample; and    -   (e) separating the T cells specific for said antigen of interest        from said complex.

In this embodiment, the solid support is preferably a glass bead ormagnetic bead of about 25 to 300 μm in diameter. Additionally, the MHC,accessory, and functional molecules are not covalently bound directly tothe solid support but are noncovalently associated with the lipid layerhaving the capacity to migrate on the surface of the solid support.Moreover, in a preferred embodiment of this example, the molecule ofinterest (e.g., MHC, accessory and functional molecules) are connectedto cholera toxin β subunit. Further, GM-1 is incorporated into the lipidlayer matrix providing a means by which the cholera toxin portion can bebound and the molecule of interest properly oriented in the lipid layer.

In still another aspect, a spheroid solid support may comprise lipidmonolayer coating the solid support with only a irrelevant moleculehaving affinity for bonding another irrelevant molecule located on anAPC that either does not have a solid support interior or does have asolid support interior. This aspect adds versatility to the captureprocess for isolation of APCs that are bound to antigen-specific Tcells.

In a further preferred aspect of the invention, kits are provided forthe isolation of T cells specific for an antigen of interest comprisingany of the following: solid supports, phospholipids, antigen-specificartificial APCs, TCR-specific artificial APCs, solid supports containinglipid associated capture molecules for capturing irrelevant molecules,solid supports containing capture molecules bound to the solid support,buffers, media, labels and column devices.

In another embodiment, the invention contemplates a method ofcharacterizing the functional state of antigen-specific T cellscomprising:

-   -   (a) isolating T cells;    -   (b) extracting mRNA from said isolated T cells;    -   (c) obtaining cDNA corresponding to said extracted mRNA;    -   (d) evaluating the mRNA encoding proteins that govern function        and phenotype of the antigen-specific T cells wherein the        evaluation is carried out by a method selected from the group        consisting of (1) mRNA translation of the proteins and testing        such proteins using antibodies against the proteins, and (2)        rtPCR of the mRNA using primers specific for the proteins.

In this method, the evaluation of the mRNA encoding proteins that governfunction and phenotype of the antigen-specific T cell may be used todetermine efficacy of an immunomodulation treatment regimen such as theadministering of a vaccine. The immunomodulation treatment can compriseinducing tolerance in autoimmunity, reducing allergic response, inducingan immune response against cancer cells. Additionally, the proteins thatgovern function and phenotype of the antigen-specific T cells includecytokines, chemokines, chemokine receptors, and cytokine receptors.

The genes encoding antigens may also be identified. In another aspect,the invention contemplates a method for identifying a gene which isexpressed by a T cell specific for an antigen of interest comprising:

-   -   (a) obtaining a biological sample containing T cells which are        specific for an antigen of interest;    -   (b) labeling with a first label at least the intracellular gene        product of interest produced by T cells in said biological        sample;    -   (c) preparing a liposome:MHC:antigen complex, wherein the        antigen in said liposome:MHC:antigen complex is said antigen of        interest;    -   (d) contacting the biological sample obtained in step (a), as        labeled in accordance with step (b), with the liposome:MHC:        antigen complex obtained in step (c) to form a        liposome:MHC:antigen:T cell complex;    -   (e) labeling with a second label the liposome:MHC:antigen:T cell        complex obtained in step (d); and    -   (f) discriminating, according to antigen specificity, cells        producing the intracellular gene product of interest, which        cells have both the first label and the second label.

In such method, the first and said second label may be selected from thegroup consisting of biotin, a flurochrome, FITC, and a radioactivelabel; provided that the first and second labels are not the same.

In yet another preferred embodiment, methods are provided for modulatingT cell responses. In this embodiment, T cells are isolated from asubject which are specific for an antigen of interest followed bycombining said isolated T cells with an artificial APC having functionalmolecules specific for modulating a T cell. In a preferred embodiment,the T cells specific for an antigen of interest are isolated using anyof the T cell isolation methods described herein. In one preferredembodiment, modulation to activate T cells generally is caused bycontacting said T cells with an artificial APC that expresses theco-stimulatory molecule B7 or anti-CD28, as well as MHC:antigen.

By “modulating T cell response” is meant the intentional intervention inthe functional characteristics of antigen-specific T cells, including,but not limited to, functional pattern of cytokines produced, change inphenotype of T cells, and modulation in the expression of activationmarkers, cytokines and their receptors and chemokines and theirreceptors. Such modulations can be carried out for numerous purposes asdiscussed in the numerous examples above.

The modulation of T cell response may further comprise changing in wholeor in part the functional pattern of cytokine production by the isolatedT cells specific for a given antigen from a Th1 response to a Th2response. In a preferred embodiment, modification of a T cell responsefor the purpose of increasing its Th2 response and/or decreasing its Th1response includes the expression by the artificial antigen presentingcell used in such method of a co-stimulatory molecule, such as B7-2.

In another embodiment, the modulation of T cell response may comprisechanging in whole or in part the functional pattern of cytokineproduction by said isolated T cells from a Th2 response to a Th1response. Preferably, modulation of a T cell response for the purpose ofincreasing its Th1 response and/or decreasing its Th2 response includesthe expression by the artificial APC used in such method of aco-stimulatory molecule such as B7-1.

The two subsets of CD4 T cells represented by Th1 and Th2 cytokinemarkers, have very different functions from one another. These two CD4 Tcell subsets can also regulate each other. Once one subset becomesdominant, it is often difficult to shift the response of the T cell(i.e. expression of the cytokine) to the other type. One reason for thisis that expressed cytokines of one type of CD4 T cell will inhibit theactivation of the T cell to expression of another cytokine. For example,IL-10, a product of Th2 cells, can help to inhibit the development of aTh1 response. Therefore, in one embodiment, IL-10 is incorporated intoartificial APCs and the resulting APC may be used to inhibit developmentof Th1 response.

In another example, interferon-γ (INF γ), a product of Th1 cells, canhelp to prevent the activation of a Th2 response. If a particular CD4 Tcell type is activated preferentially in a response so that the cytokine(Th1 or 2) is highly expressed, such high expression can suppress thedevelopment of the other subset. The overall effect of T cellpopulations expressing one or the other subset is that various tissuesbecome either suppressive (Th2) or inflammatory (Th1). Thus, INF γ canbe incorporated into artificial APCs and the resulting APC used tomodulate cell populations to become either suppressive or inflammatory.

The ability to manipulate T cell response (as exemplified by the Th1/Th2subsets) provides a novel method by which treatment can be provided fornumerous disease states. For example, a response to an allergen can bemanipulated so as to shift the antibody response away from anIgE-dominated response. Such a shift will prevent the allergen fromactivating IgE-mediated effector pathways. For example, a technique thathas been used for many years to generate a desensitization response iscarried out by contacting patients with escalating doses of the allergen(such as by feeding, nasal delivery, or by injection). This immunizationschedule appears to gradually divert an IgE-dominated response, whichdiversion is driven by Th2 cells, to one driven by Th1 cells, with theconsequent down-regulation of IgE production. Thus, the activation of aTh2 response in such a manner may be of use in the treatment of allergy.

Similarly, other conditions which are associated with a Th2 T cellresponse, as for example, responses in which the functional phenotype ofcytokine secretion is tolerogenic to viral infections and some types ofcancer, may also benefit from a modification of the Th2 T cell responseso that Th2 is reduced and/or a Th1 T cell response is increased.

Autoimmune conditions, in which the functional phenotype of cytokinesecretion is pro-inflammatory (as is the case with Th1), will benefitfrom a modification of the Th1 T cell response so that it is reducedand/or a Th2 T cell response is increased. As noted above, cytokines arecell-derived soluble mediators associated with immune responses that mayact both within a microenvironment and/or systemically. Immune cellssecrete specific cytokine profiles, each having markedly differenteffects. Th2-type cells secrete low levels of TGF β, no IFN γ and highlevels of IL-4 and IL-10, the presence of which in turn produce animmunosuppressive or tolerogenic immune environment. Th1-type cells onthe other hand secrete very low TGF β, high IFN γ and no IL-4 or IL-10.High expression of INF γ is associated with cell mediatedproinflammatory immune environments. As discussed herein, the artificialAPC of the current invention can be used to modulate T cell responses byaffecting these and other cytokines. Moreover, such cytokines as well asother soluble factors to which a T cell responds may be used inconjunction with a column device as described below.

In yet another embodiment, the regulation of T cell responses maycomprise inducing anergy. Specifically, T cell responses may be modifiedfor the purpose of inducing anergy through the Fas/Fas Ligand pathway.

In yet another aspect, the present invention provides methods oftreating a condition in a subject who would be benefited by modulatingthe functional patterns of cytokine production by certain of suchsubject's antigen-specific T cells to increase Th2 response and/ordecrease Th1 response, comprising:

-   -   (a) isolating T cells capable of triggering a Th1 response from        a subject;    -   combining said isolated T cells with artificial APCs which        express MHC capable of binding an antigen recognized by said T        cells wherein said artificial APC also expresses the        co-stimulatory molecule B7-2;    -   (c) separating T cells that have bound to said artificial APCs        in step (b); and    -   (d) administering said T cells isolated in step (c) to said        subject.

T cells that have been in contact with an artificial APC as describedabove will be stimulated to shift their Th1 response to a Th2 response.Conditions which would be benefited by modulating the functional patternof cytokine production to increase Th2 response and/or decrease Th1response include autoimmune diseases such as, for example, type 1diabetes mellitus, multiple sclerosis, rheumatoid arthritis,dermatomiosytis, juvenile rheumatoid arthritis and uveitis.

In yet another aspect, the present invention provides methods oftreating a condition in a subject that would be benefited by alteringthe functional pattern of cytokine production by certainantigen-specific T cells to increase Th1 response and/or decrease Th2response comprising:

-   -   (a) isolating T cells that are specific for an antigen capable        of triggering a Th2 response from a subject;    -   (b) combining said isolated T cells with an artificial APC which        expresses an MHC capable of binding an antigen that is        recognized by said T cells so that the functional pattern of        said T cells may be modulated, wherein said artificial APC also        expresses the co-stimulatory molecule B7-1;    -   (c) separating the T cells modulated in step (b); and    -   (d) administering said T cells isolated in step (c) to said        subject.

Conditions which would be benefited by modulating the functional patternof cytokine production to increase Th1 response and/or decrease Th2response include for example, allergy, allergy to dust, animal skinbypass products, vegetables, fruits, pollen, chemicals, and someinfections (e.g., viral, fungal, protozoan and bacterial).

As noted above, the present invention provides methods for isolatingantigen-specific T cells. It is desirable to isolate antigen-specific Tcells in a variety of contexts. For example, in adoptive immunotherapy,lymphocytes are removed from a patient, expanded ex vivo, and thenreinfused back into the patient to augment the patient's immuneresponse. See Rosenberg et al., N. Engl. J. Med. 313:1485–1492 (1985);U.S. Pat. No. 4,690,915. This approach has been effective in thetreatment of various cancers (see, e.g., Rosenberg et al., N. Engl J.Med. 319:1676–1680 (1988). Isolation and expansion of T cells specificfor a particular antigen will increase the specificity and effectivenessof adoptive immunotherapeutic approaches. Such expansion may bebenefited by obtaining a population of monoclonal T cells. Thus, theinvention contemplates a method of obtaining a monoclonal population ofT cells specific for an antigen of interest comprising:

-   -   (a) isolating T cells specific for an antigen of interest; and    -   (b) culturing the isolated T cells in an individual well with        the antigen of interest and an artificial APC.

Isolated T cells specific for a particular antigen may also be used as adiagnostic to screen for the presence of, or amount of a particularantigen and thus detect the presence, absence, or status of an immuneresponse. Early detection of an immune response will facilitateselection of a particular treatment regimen in a variety of pathologicalconditions such as autoimmune diseases, allergies, allograft rejection,and infectious diseases. In a diagnosis of this type, the isolated Tcells are used both as a means of detection and as reporters. The Tcells proliferate when contacted with the antigen for which they arespecific. This proliferation is easily detected as an increase in cellnumber or as an increase in growth rate measured, for example, by therate of uptake of a label (e.g., observation of the uptake of tritiatedthymidine or bromodeoxyuricyl (BrdU)). Thus, the presence or absence oftarget antigen can be detected by exposing the isolated T cells to atissue sample (e.g., peripheral blood) and monitoring theirproliferation rate. In the current invention, a preferred embodimentincludes the observation of bound T cells using a FACS which allows theavoidance of time consuming proliferation experiments.

Isolation of antigen-specific T cells also provides a homogenous sourceof T cell receptors. A homogenous source of T cell receptors is an aidto the elucidation of structure-function relationships of particularreceptors. A homogenous source of T cell receptors also facilitates thedevelopment of solubilized T cell receptors that are of use in a numberof therapeutic applications (See, e.g., U.S. Pat. No. 5,283,058). In thecurrent invention, solubilized receptors may be used in conjunction withan immunomodulation column device.

In another preferred embodiment, the invention provides for a method ofidentifying epitopes expressed on the MHC that are of import toacceptance or rejection of grafts in transplantation therapy. In thisembodiment, a donor's MHC is examined by computer modeling to identifypeptide moieties likely to be recognized by a recipient's T cells. Therecipient's T cells are tested by FACS analysis for binding against thepeptides. Upon positively identifying reactive peptides, such peptidesmay then be used to deplete the recipient's graft rejecting T cellsthereby modulating the recipient's immune response in thetransplantation regimen. This modulation is carried out within acomprehensive treatment that includes further modulating the recipient'ssensitivity to the graft epitopes by exposing the recipient to thedonor's epitopes by feeding, nasal delivery, or injection of increasingconcentrations of the peptides.

In another aspect, the invention provides for a method to identifyantigenic motifs of pathogens that are recognized by the MHC. In thisembodiment the identification of such motifs allows the development ofanti-pathogen vaccines comprising either pools of such motifs in theform of peptides that are recognized by T cells in the general humanpopulation i.e., the pathogen's antigenic moieties responsible forgenerating immune responses are identified so that for any disease, avaccine comprising such antigens may be produced. Additionally, avaccine may be produced against such antigenic moieties comprising apopulation of a patient's pathogen-specific T cells that have beenexpanded ex vivo.

In yet another aspect of the invention, a column device is providedcomprising a multiplicity of compartments that are arranged in series.As shown in FIG. 8, one embodiment of such device has compartments A, B,and C. Each compartment is contiguous with the adjacent compartment by avalve 1. The valve between each compartment may be opened or closed.Compartment A also has entrance ports 2 and exit ports 3 for receiving aflowable medium. Any of the compartments may contain solid supports asseen in FIGS. 6, and 7 for binding either a molecule that can bind to anirrelevant molecule for immobilizing an artificial APC, or for bindingdirectly MHC:antigen:functional molecule complexes. The entrance port 4of compartment A is attached to an external device 5 (e.g., a devicewhere a patient's T cells are separated from whole blood andconcentrated) prior to export through a sterile tube 6 to entrance port4. Once the patient's enriched T cell population is transported intocompartment A via entrance port 4, the T cells containing TCR specificfor the MHC:antigen complexes bind to MHC:antigen complexes that arethemselves attached either directly to the solid supports or areassociated with an artificial APC which is bound to a solid support viaan irrelevant molecule. In the configuration demonstrated in FIG. 8,artificial APCs 14 are attached to solid supports in compartment βawaiting interaction with incoming cell through entrance port 4.Compartment A thus provides a chamber wherein the APCs and cells caninteract so that antigen-specific T cells can be isolated whilenon-binding T cells are allowed to wash out of the compartment A viaexit port 3 a. After the non-binding cells are removed (and eitherdiscarded or returned to the patient's circulatory system, or addressedto a device for monitoring the cells such as a FACS), the boundantigen-specific T cells may be released from the solid supports in Aand allowed to channel or flow through valve 1 located betweencompartment A and B and into compartment B. The artificial APC-cellbound complexes or just the antigen-specific T cells alone may bereleased from solid support of compartment A by adjusting thetemperature of the medium in the compartment A. Once theantigen-specific T cells are in compartment B, they may again becaptured as shown in FIG. 8, or simply be treated without recapture inany number of ways to induce cell modulation. For example, artificialAPCs containing antigen-specific MHC:antigen:functional moleculecomplexes may be infused into compartment B via entrance port 7. Withthe infusion of the antigen-specific APCs into compartment B, theantigen-specific T cells may then bind and be induced to modulate theirrespective response or be acted upon otherwise as desired. FIG. 8 showsT cells 13 that have been released from compartment A and are shownbound to APCs that are in turn bound to solid supports. As describedabove, and hereinafter, numerous types of modulation of the T cells mayoccur. For example the T cells may be modulated to (1) increase a Th 1and/or decrease a Th2 response, (2) increase a Th2 and/or decrease andTh1 response, (3) be generally induced to proliferate regardless of Th1or Th2 response, (4) enter a state of anergy, (5) become apoptotic, (6)shed certain adhesion molecules known to those skilled in the art to beinvolved in the entry of cells into specific diseased areas, (7)upregulate/downregulate chemokine or cytokine receptors associated witha Th1-type response, (8) upregulate/downregulate chemokine or cytokinereceptors associated with a Th2-type response. The T cells may also becaptured on such APCs for the purpose of (1) depletingallo-antigen-specific T cells from a donor's T cell population intransplantation oriented therapy, such as bone marrow transplant therapyor (2) from a patient's T cell population such as to facilitate a graftof allogenic solid organs, or (3) used in identifying TCRs that bind topathogen-recognizing MHCs or self-derived pathogen-mimic antigenicmotifs. Moreover, soluble molecules may be added to the device forinducing such modulation including cytokines, adjuvants, and hormones.

Once isolation or other modulation has been performed in compartment B,the T cell:artificial APC complex may be addressed to compartment C viathe valve 1 between compartments B and C. The entrance port 7 ofcompartment B may be additionally connected to external units forsupplying buffers and the like that may be necessary for carrying outmanipulations of the T cells in compartment B. The exit port 8 ofcompartment B may also be used to transport modulated T cell:artificialAPC complexes to sampling devices such as FACS or to other devices forsuch things as cell proliferation and the like.

Compartment C may also contain solid supports and the like for bindingartificial APC:T cell complexes and further treatment. In compartment C,the modulated and isolated T cells may be eluted from the APCs (via theadjustment of the medium's temperature) and addressed to other devicesthrough ports 9 and 10, or addressed out of the column through exit port11 to, for example, the patient, a FACS, a culture device, or to anotherlocation for further manipulation.

Specific embodiments of the present invention are exemplified in thefollowing Examples. These Examples are not to be interpreted as limitingthe scope of the invention in any way, the scope being disclosed in theentire specification and claims.

EXAMPLE 1 Liposome Assay for Detection of Antigen-Specific T Cells

In this example, experiments are described which demonstrate thecapacity of T cells to bind to liposomes containing cholesterol havingMHC:antigen complexes inserted into the liposome membrane. The capacityof T cell binding was quantified by flow cytometry analysis (FACS).Negative controls for the binding include the use of a control T cellline (i.e., non-reactive) having specificity for an irrelevant peptide,incorrect MHC restriction, peptide and antibody inhibitions, limitingdilutions, and the use of MHC without peptide.

The ability of the method to provide for discrimination betweenantigen-specific T cells was facilitated by use of two T cell hybridomasspecific for the same peptide. These hybridomas were OVA³²³⁻³³⁶ (whichcorrespond to residues 323–326 of ovalbumin) (obtained from ResearchGenetics, Huntsville Ala.) which were restricted by two different MHCs,I-A^(S) and I-A^(d). Specifically, the designations for the restrictionswere I-A^(S) restricted OVA³²³⁻³³⁶ specific T cell hybridoma, AG111.207,and the I-A^(d) restricted OVA³²³⁻³³⁶ specific T cell hybridoma8D051.15. A peptide containing 2 identities and one conservativesubstitution, Hi 15 (Research Genetics), which corresponds to residues15–31 of H. influenzae isoleucyl tRNA transferase, was used as anegative control.

Materials and Methods

Preparation of Liposome:MHC:Antigen Complexes.

Liposomes were prepared similarly to that described by Brian et al,PNAS, 81:6159–63. Briefly, cholesterol (Ch) and L-α-phosphotidylcholine(PC) (Sigma) were mixed at a molar ratio of 2:7 of Ch and PC,respectively. The mixture was placed under an argon stream for 30minutes to evaporate chloroform used in the preparation, and resuspendedin 140 mM NaCl, 10 mM Tris HCl, and 0.5% deoxycholate at pH 8. Thesuspension was sonicated for three minutes or until clear.

Complexes of affinity-purified MHC molecules I-A^(S) and I-A^(d) (eachexpressed in a B cell lymphoma and purified via immunoaffinity column)were inserted into liposomes by a 72 hour 4° C. dialysis against threechanges of PBS (Slidalyzer, Pierce) at a 1:10 molar ratio of MHC toliposomes to form liposome:MHC complexes.

The OVA³²³⁻³²⁶ peptide and the control peptide, Hi 15, werebiotinylated, (post synthesis, Sigma), and the biotinylated peptides(b-peptides) were incubated with the liposome:MHC complexes for 18 hoursat room temperature at a physiologic pH to form liposome:MHC:b-peptidecomplexes.

Flow Cytometry.

Viable cells were separated from debris using a ficol-hypaque gradient.(Lymphocyte M) Cells were blocked with 10% PCS in PBS for 10 minutes onice, then washed in PBS. Incubations with antibodies (used atconcentrations of 400–600 ng/ml) were performed on ice in the dark for20 minutes. The antibodies used included anti-CD3e (clone 145-2C11),anti-CD4 (clone GK 1.5), anti-CD8a (clone 53-6.7), anti-HSA (cloneM1/69), and anti-CD69 (H1.2F3) (Pharmingen, San Diego, Calif.).Liposome:MHC:b-peptide complexes were preincubated with fluorescentstreptavidin molecule (f-strep) at room temperature for ½ hour. When twopeptides were used in the sarne assay, the liposome:MHC:b-peptidecomplex was preincubated with streptavidin molecules of differingfluorescence prior to addition to cells. Sorted cells were either (a)cultured (using a 3:1 ratio of irradiated BALB/c spleen cells, 20 U/mlrIL-2, 10 μg/ml Hi 15 or Iα52, 10% FCS, 1%Penicillin/Streptomycin/Glutamine (P/S/G), in RPMI 1640 at 37° C. and 6%CO₂), (b) processed for variable beta chain (Vb) analysis by PCR, or (c)reanalyzed by a fluorescent antibody cell sorter (FACS).

Yields ranged from 2,000 to 16,000 events. Bulk-sorted cells used forreanalysis were incubated for ½ hour on ice and spun down through 100%FCS at 325×g for 10 minutes to remove liposome:MHC:b-peptide complexes,prior to restaining with liposome:MHC and a different b-peptide.Single-cell sorts were dispersed in 96-well culture plates containingfresh irradiated APCs obtained from the spleen of a syngeneic BALB/cmouse. Generally 8–12 wells showed proliferation over six weeks. Cellswere visualized with a Becton Dickinson FACS Star equipped with LYSIS IIsoftware.

As shown in FIG. 9, specific recognition of MHC/peptide complexes by T—Thybridomas AG111.207 (I-A^(S)/OVA³²³⁻³³⁶ specific) and 8D051.15(IA^(d)/OVA³²³⁻³³⁶ specific) were observed. Cultured AG111.207 or8D051.15 cells were analyzed by flow cytometry using anti-CD4 antibodiesand I-A^(d)% b-peptide or IA^(s)/b-peptide combinations complexed intoliposomes and visualized by addition of f-strep. The ratio of cellsspecific to either the I-A^(d)/antigen or A^(s)/antigen was constantbetween experiments. (All figures are gated on CD4+ cells unlessotherwise stated.) FIG. 9( a) shows AG111.207 cells which were stainedwith anti-CD4 antibody and differing combinations of MHC/antigenincluding an irrelevant peptide known to bind to I-A^(d) (Hi 15).Cellular specificity is shown by use of a B cell hybridoma (FIG. 9-a 4).FIG. 9( b) shows 8D051.15 cells which were analyzed with differingcombinations of MHC:antigen. Included is a control using biotinylatedI-A^(d) alone to detect the T cell hybridoma (FIG. 9-b 4).

Results

In the series of experiments for which results are shown in FIG. 9,purified I-A^(S) or I-A^(d) MHCs were inserted into liposomes and thencomplexed to the biotinylated OVA³²³⁻³³⁶ peptide. These complexes wereincubated with streptavidin-FITC, and then with a standard amount ofAG111.207 or 8D051.15 cells (final MHC concentration of 66 μg/ml). Whenanalyzed by flow cytometry, nearly 90% of the AG111.207 (FIG. 9 a 1) and87.2% of 8D051.15 (FIG. 9 b 1) cells stained positive when using thecorrect restriction and peptide.

The specificity of the entire interaction was demonstrated by lack ofstaining of AG 111.207 and 8D051.15 cells when incubated with anti-CD4Ab and complexes of the incorrect restriction for each hybridoma,I-A^(d) and I-A^(S) respectively, and Hi 15 which has two identities(p2, p10) and one conservative substitution (p5) with OVA³²³⁻³³⁶ (0%positive cells FIG. 9 a 2; 2.5% positive cells, FIG. 9 b 2). The peptidespecificity of the interaction was demonstrated when AG111.207 and8D051.15 cells were incubated with anti-CD4 antibody, the correctrestriction element, but irrelevant peptide for each hybridoma,I-A^(s)/b-Hi15 or I-A^(d)/b-Hi15, respectively (4.6% positive cells,FIG. 9 a 3; 8.7% positive cells, FIG. 9 b 3). The binding betweenMHC:b-peptide complexes and AG 111.207 T cells was also concentrationdependent. Only 13.1% of AG 111.207 cells tested positive when theI-A^(s)OVA³²³⁻³³⁶ concentration in the assay was reduced five fold to 13μg/ml (not shown). The signal was also reduced by the addition of 300μg/ml of the same, non-biotinylated OVA³²³⁻³³⁶ peptide as a competitiveinhibitor during preparation of the I-A^(s)/OVA complexes (5.1% of CD4+cells positive, not shown). This finding suggests that biotinylation ofthe peptide does not interfere with the trimolecular interactions amongpeptide, MHC and TCR. Moreover, this assay was seen to be dependent onboth TCR and MHC:peptide complexes, insofar as binding can be inhibitedby simultaneous addition of anti-TCR and anti-I-A antibodies (6.9% ofCD4⁺ cells positive). As shown in FIG. 9 a 4, no binding to TCR-negativecells, such as B cell hybridoma HT.01, was detected (0% of cellspositive). Using biotinylated I-A^(d) in liposomes without peptide, 6.9%of 8D051.15 cells bound the MHC alone (FIG. 9 b 4). Hence, theinteraction requires the presence of the specific peptide.

Artificial APCs Identify T Cell Hybridoma 8DO Specific for IA^(d)/OVACombination

In another experiment we evaluated the capability of artificial APC,presenting synthetic biotynilated peptide OVA in the context of IA d, tovisualize by FACS analysis hybridoma 8D0, which is OVA/IA^(d) specific.As shown in FIG. 15, the percentage of hybridoma cells was visualized bybinding with cychrome-tagged artificial APC. This interaction wasspecific, insofar as TCR binding was dependent on the availability ofthe MHC/peptide complexes. The interaction was inhibitable by additionof antibodies interfering with such interaction (% positive cells, FIG.15 middle graph), and 8D0 hybridoma cells did not bind to the artificialAPC presenting the correct peptide in the context of IE^(d) (% positivecells, FIG. 15 left graph). The result indicates that highly specificand sensitive interaction occurs between T cells and artificial APCs.Moreover, addition of competitive inhibitors in reducing the specificbinding proves that the labeling of this method does not interfere withthe APC/T cell interaction.

EXAMPLE 2 Identification of Antigen Specific T Cells in Mouse EmbrionicThymuses

In this example, a murine model is used to investigate the effects of anaturally processed self-peptide on the maintenance and proliferation ofT cells which may cross-react with homologous peptides of exogenousorigin, all performed in a non-transgenic system. The example emphasizesthe fact that without a method such as that of the current invention tocapture T cells, it is not possible to evaluate polyclonal antigenspecific chimeric T cell selection in a non-transgenic system. This isbecause conventional methods that examine cytokine presence or cellproliferation cannot identify cells specific for relevant antigen.

The self-peptide I α 52 (ASFEAQGALANIAVDKA) (Seq. Id. No. 1), used inthese experiments corresponds to residues 52 to 68 of the α chain of theI-E molecule. It represents one of the most abundant peptides naturallyprocessed and presented in the context of I-A^(d) (Hunt et al., Science,Vol. 256:1817–20; 1992; Rudensky et al., Nature, Vol. 353:622–27; 1991).These experiments demonstrate that T cells specific for a particularantigen can be identified from a polymorphic population of cells. Inaddition, functional and genetic characteristics of the cell populationwere demonstrated (Table 1).

TABLE I Phenotypic and Functional Characteristics of Thymocytes Beforeand After Fetal Thymic Organ Culture Day 6 of Day 16 FTOC⁺ of Day 6 of10⁻² mM Gestation FTOC Iα52 CD3^(lo)CD4⁺CD8⁺  6%  1.3%  1.1%CD3^(lo)HSA^(hi) 99  3.9  4.1 CD3^(lo)CD69⁺  2 <1 <1 CD3^(hi) <1 47 34CD4+CD8+ CD3^(hi)HSA^(hi) <1 82 84 CD3^(hi)CD69⁺ <1 10 11 Cell NumberN/D  1.55 × 10^(5#)  1.65 × 10^(5#) Iα52 Stimulation N/D  0.5 × ⁺/−0.1 6.2 × ⁺/−2.7 Index Hi15 Stimulation N/D  0.1 × ⁺/−0.06  2.5 × ⁺/−1.1Index BALB/c embryonic thymus lobes were made into single cellsuspensions, and analyzed by flow cytometry. Cell numbers are an averageof eight to ten thymus lobes from FTOC. Data in the table representbetween two and four experiments. ^(#)Standard deviations are 3.1 × 10⁴and 1.5 × 10⁴ respectively.BALB/c embryonic thymus lobes were made into single cell suspensions,and analyzed by flow cytometry. Cell numbers are an average of eight toten thymus lobes from FTOC. Data in the table represent between two andfour experiments. #Standard deviations are 3.1×10⁴ and 1.5×10⁴respectively.

Materials and Methods

Antigens.

The I-A^(d)-derived peptide Iα52 (ASFEAQGALANIAVDKA) (Seq. Id. No. 1)was synthesized (Research Genetics) using standard solid phase peptidesynthesis method. Peptides used for flow cytometry were biotinylated(b-peptides) using a kit (Sigma) and were separated from free biotin byHPLC.

Generation of Lymphocytes from Fetal Thymic Organ Cultures (FTOC).

BALB/c embryonic thymi were harvested day 16 of gestation and placedonto 0.4 μm cell culture inserts (Fisher) in six-well plates containingRPMI 1640, 10% FCS, 1% P/S/G, with or without Iα52 (10⁻¹ mM to 10⁻¹⁰mM), at 37° C. and 6% CO₂ for six days. Cells were dissociated with aglass grinder (Kontes). Cell recovery was between 1.0×10⁵ and 2.8×10⁵cells/lobe. Post-FTOC cultures were performed using a 3:1 ratio ofirradiated BALB/c spleen cells, 20 U/ml rIL-2, 10 μg/ml Hi 15 or Iα52,10% FCS, 1% P/S/G, in RPMI 1640 at 37° C. and 6% CO₂.

Preparation of Liposome:MHC:Antigen Complexes.

Liposomes were prepared as described in Example 1.

Flow Cytometry.

Flow cytometry was performed as described in Example 1.

Results

It has been shown previously that different concentrations of the samepeptide have strong implications for T cell selection (see Sebzda etal., Science, Vol. 263:1615–18). We confirmed this phenomenon usingvarious concentrations of Iα52 peptide to demonstrate its influence uponpeptide-mediated positive selection and to define, directly, the antigenspecificity of a positively selected T cell population in anon-transgenic BALB/c model.

FIG. 10 shows the influence of Iα52 on the maturation of antigenspecific thymocytes in fetal thymic organ cultures. FIG. 10( a)represents differing concentrations of Iα52 added to 6-day FTOC usingday 16 of gestation BALB/c embryonic thymus lobes. Thymocytes wereanalyzed by three color flow cytometry to determine the percentages ofCD3HI, CD4, and CD8 double positive and CD3HI CD4 single positive cells.With FTOC alone in the absence of any added peptide, 20.7% of CD4⁺ cellsbound liposome:I-A^(d):biotinylated-Iα52 complexes during one week. Thisfinding reflects the availability of Iα52 as an abundantly represented,naturally processed peptide available for thymic selection.

FIG. 10( b) shows VP analysis by FACS of cells taken directly from day16 of gestation embryonic thymus lobes, and from FTOC supplemented with10⁻² mM Iα52.

FIG. 10( c 1) represents thymocytes from FTOC without antigen, or withaddition of the Iα52 peptide (c 2), which were analyzed by FACS usingpropidium iodide to measure cell viability. Analysis of CD4, CD8 ratiosfrom the two cell cultures (c3,4) is representative of those used in theabove titration.

Performance of the detection of antigen specific thymocytes by FACSusing anti-CD4 antibody and liposome:I-A^(d):b-Iα52 complexes bound tof-strep is shown in FIG. 11. When thymic lobes were cultured for oneweek without the presence of 10⁻² mM Iα52 (FIGS. 11-1), a relativeincrease (to 50.9%, FIGS. 11-2) ofliposome:I-A^(d):biotinylated-Iα52.TCD4⁺ cells was seen in the presenceof 10⁻² mM Iα52. These data demonstrate for the first time that aself-derived peptide can induce positive selection in a non-transgenicsystem. As a control for antigen specificity in the T cell captureassay, the unrelated, biotinylated peptide EBV1 (TRDDAEYLLGRESVL), (Seq.Id. No. 2) derived from the EBV protein balf2 (residues 1030–1045) wasused. EBV1 binds efficiently to I-A^(d), and is a good immunogen inadult BALB/c mice (La Cava et al, submitted). Biotinylated EBV1 peptidecomplexed to I-A^(d) in liposomes bound 0% of CD4+ cells from FTOC (FIG.11-3).

EXAMPLE 3 Identification of Cross Reactive T Cells With Specificity forHomologous Peptides

The method of the invention was used to further examine the capacity forclosely related antigenic moieties to cross-react, in this case usingthe murine model seen in Examples 1 and 2. Evidence of suchcross-reaction provides the basis for an explanation of some autoimmunedisease states. In this Example, the ability of T cells selected by theself-MHC-derived peptide Iα52 to cross-react with the homologous peptideof non-self origin Hi15, was examined by performing antigen-specific Tcell analysis.

Materials and Methods

Antigens.

The I-A^(d) derived peptide Ioc52 was synthesized as described inExample 2. Hi15 (TSFPMRGDLAKREPDK) (Seq. Id. No. 3) was synthesized bystandard solid phase peptide synthesis technique (Research Genetics). Hi15 was identified among 20 candidates with an arbitrary homology scoreof 20, based on homologies including potential MHC-binding residues. Thesearch was performed on the non-redundant database scanned by Blast 2program, available on the NCBI website. Peptides were >90% pure(Research Genetics). Peptides used for flow cytometry were biotinylated(b-peptides) using a commercial kit (Sigma) and were separated from freebiotin by HPLC. Experiments were performed to determine the possibleinterference of the biotin label en the specificity of T cell receptorsfor the Hi15 peptide using the post-synthesis biotinylated Hi 15 andN-terminus biotinylated peptide separately complexed to MHC molecules inliposomes and labeled with different colors of conjugation tostreptavidin molecules. Results showed equal binding of the twopreparations to a T cell line having specificity for both Iα52, and Hi15 (not shown).

Generation of Lymphocytes from Fetal Thymic Organ Cultures (FTOC).

FTOC was prepared as described in Example 2.

Preparation of Liposome:MHC:antigen Complexes.

Liposomes were prepared as described in Example 1.

Flow Cytometry.

Flow cytometry was performed as described in Example 1.

Results

These experiments show that a T cell population derived fromIα52-supplemented FTOC can be determined, characterized and expanded.Essentially, an artificial APC will comprise MHC:peptide complexesstabilized into liposomes, with the addition of transmembrane proteinswhich accomplish stabilizing, co-stimulatory, and/ormodulator/functions. These accessory and co-stimulatory moleculescomprise, but are not limited to, any or all of the following:

-   -   (i) ICAMI as adhesion molecule to facilitate initial interaction        between the T cell and the APC.    -   (ii) anti-CD28 transmembrane antibody facilitates the        propagation of antigen-specific T cells isolated by T cell        capture using artificial APCs such that up to 20 replicative        cycles have been obtained, which represents a valid alternative        to T cell expansion and cloning using autologous APC systems,        often an insurmountable hurdle in human systems.    -   (iii) B7-1 may be used instead of anti-CD28. The cells obtained        from this treatment may exert immunoregulatory function in        autoimmunity.    -   (iv) B7-2 may be used instead of anti-CD28. Cells obtained from        this treatment may have immunomodulatory properties in settings        such as cancer or infectious disease.

All of the aforementioned molecules are transmembrane proteins which canbe incorporated into liposomes according to above examples, or as analternative, bound to a solid support as in above examples.

Isolation and immunomodulation of antigen specific T cells.

Antigen-specific T cells isolated according to above examples areincubated with the appropriate variant of the artificial antigenpresenting cells according to the desired objective, i.e. expansion,functional phenotype switch, etc. Day 16 of gestation BALB/c embryonicthymus lobes were harvested and cultured on 0.4 μm filters in mediumsupplemented with 10⁻² mM Iα52. Lobes were dissociated and cultured withautologous, irradiated APC, IL-2 and the Hi 15 peptide. After two weeksof incubation with 10μg/ml of Hi15, FACS analysis showed that 52.8% ofcells bound anti-CD4 and the self-derived I-A^(d):Icx52 peptidecomplexes (FIGS. 12-1). The liposome:I-A^(d):biotinylated Iα52⁺:CD4⁺cell complexes were sorted and depleted of their liposome:I-A^(d):Iα52⁺components by incubation at 4° C. for thirty minutes followed bycentrifugation at 325×g for 10 minutes through 100% PCS (FIGS. 12-2).The sorted cells were then restained with liposome:I-A^(d) and theexogenous, biotinylated peptide Hi 15 which was complexed to astreptavidin molecule conjugated to a fluorochrome. The restained Tcells were then reanalyzed by three-color flow cytometry using anti-CD4antibody, liposome:I-A^(d):b-Hi15 complexes, and liposome:I-A^(d):b-Iα52complexes bound to different color f-strep molecules. Results showedthat 52% of the cells tested positive (FIGS. 12-3). To furtherdemonstrate the ability of TCRs selected by Iα52 to recognize theexogenous antigen Hi 15, T cell populations derived from Iα52 FTOC wereexpanded with Hi 15 and incubated with anti-CD4 antibodies andlipsome:I-A^(d) complexes bearing either biotinylated-Iα52 or Hi15 boundto streptavidin molecules labeled with different fluorochromes. Ourresults indicated that 31.1% of the CD4⁺ cells were positive for bothpeptides (double positive) (FIGS. 12-4). The results of this Example 3show that positive selection by a self-peptide generates CD4⁺ T cellsthat can recognize a homologous, exogenous peptide.

EXAMPLE 4 Identification of TCR Usage by Antigen Specific T Cells

Variable regions of TCRs on the cross-reactive cells identified inExample 3 were evaluated in order to demonstrate the absolute ability ofthe method of the current invention to evaluate TCR specificity andpotential cross-reactivity seen at a molecular level.

Materials and Methods

Antigens.

The Iα52 (ASFEAQGALANIAVDKA) (Seq. Id. No. 1) peptide (ResearchGenetics) and the Hi15 (TSFPMRGDLAKREPDK) (Seq. Id. No.3) peptide(Research Genetics) were >90% pure. Peptides used for flow cytometrywere biotinylated (b-peptides) using a commercial kit (Sigma) and wereseparated from free biotin by HPLC.

Generation of Lymphocytes from Fetal Thymic Organ Cultures (FTOC).

FTOC were prepared as described in Example 2.

Preparation of Liposome:MHC:Ag Complexes.

Liposomes:MHC:antigen complexes were prepared as described in Example 1.

Flow Cytometry.

Flow cytometry was performed as in Example 1.

Analysis of T Cell Receptor Gene Usage.

To analyze the TCR-Vβ repertoire, 500 FACS-sorted cells were washedtwice and poly (A) mRNA was isolated with the Invitrogen Micro FastTrack mRNA Isolation kit (Invitrogen, San Diego). The resulting mRNA wasquantified with DNA DipStick kit (Invitrogen) and equal amounts of mRNAfrom each source were reverse transcribed using Invitrogen's cDNA Cyclekit. The cDNA was then submitted to a first PCR by using specific Vβsense primers and a common Cβ antisense primer (Lessin, et al., J.Invest. Dermatol, 96:299–302, (1991) herein incorporated by reference).PCR conditions were 200 uM dNTPs, Ix Taq polymerase buffer, cDNA, 20 μMeach primer, 0.6 U Taq DNA polymerase (Boehringer Mannheim) in a 25 ultotal reaction volume. After 3 minute hot start at 94° C. and additionof Taq DNA polymerase at 80° C. using a Perkin Elmer 2400 thermalcycler, reactions were cycled 40 times consisting of 30 seconds at 94°C., 30 seconds at 55° C. and 40 seconds at 72° C., last extension 6minutes. Five microliters were used as template for a second PCR, whoseconditions were identical to the previous PCR, except that reactionswere cycled 35 times and 0.125 U of Taq DNA polymerase was used.Twenty-five microliters of the resulting PCR products were then analyzedby electrophoresis through a 4% agarose gel and ethidium bromidestaining. DNA sequencing was performed at The Scripps Research InstituteCore Facility using an ABI system.

Results

T cell lines obtained from either FTOC supplemented with 10⁻² mM Iα52,or from peripheral lymph nodes of animals immunized with Iα52, weresubsequently expanded in the presence of Hi 15 and IL-2. Cells weresorted by FACS, as described in Example 1 using cytometry based onanti-CD4, liposome:I-A^(d):biotinylated-Hi15 andliposome:I-A^(d):biotinylated-Iα52 triple binding. Then, RT-PCR specificfor the known TCR Vβ families was performed.

The results of these experiments showed almost exclusive use of Vβ1 bycells that recognized both the selecting self-peptide Iα52 and thecross-reactive, non-self homologue—Hi 15-(Table II). We also obtainedDNA sequences from three representative clones. This analysis showedonly modest differences in amino acid sequence for variousrepresentative clones, which all utilized Vβ1 genes (Table II).Molecular modeling showed that these amino acid sequences had littleeffect on the predicted conformation of the Vβ chain used by thedifferent clones (FIG. 9).

TABLE II CDR3 Sequence Comparison and Variable Region Gene Usage of Iα52and Hi 15 Cross-Reactive T Cell Receptors T Cell Line Isolated VDJsequences R3 F17 LHIS AVDPEDS AVYFCASSQEFFSS YEQYFGPGTRL* R3F16 I R3F15T Variable Region (Vβ) Gene Usage F7d 1, 8 Lines were generated fromsingle cell sorting by flow cytometry using the T cell capture assay.Detection in the assay was based upon I-Ad/b-Iα52 and I-Ad/b-Hi 15double binding. These sequences contain the Vβ1 region of murine TCR inpositions 78–107. *Seq. Id. No. 4.

EXAMPLE 5 Identification, Isolation and Characterization of T CellsSpecific for Rheumatoid Arthritis-Related Antigens

Initiation of autoimmune diseases such as rheumatoid arthritis isthought to be dependant on the recognition by T cells of one or more“pathogenic” antigens which may be responsible for triggeringproinflammatory events which lead to chronic autoimmune damage. Severaldifferent hypotheses and experimental approaches have led to theidentification of a pool of potential pathogenic antigens.

In this Example 5, it is demonstrated how T cells specific forrheumatoid arthritis-related antigens can the identified and enumeratedstarting from polymorphic T cell populations with very diversespecificities. Peripheral blood mononuclear cells (PBMC) from a patientwith rheumatoid arthritis are incubated with a peptide called dnaJp1(U.S. Pat. No. 5,773,570) corresponding to certain positions of the E.coli heat shock protein dnaJ. Certain peptides derived from dnaJ arehomologous to rheumatoid arthritis-associated HLA alleles, and have beenshown to be immunogenic in patients with rheumatoid arthritis. (Albaniet al., “Positive selection in autoimmunity: Abnormal immune responsesto a bacterial dnaJ antigenic determinant in patients with earlyrheumatoid arthritis,” Nat. Med. 1:448–452 (1995); Albani & Carson, “Amultistep molecular mimicry hypothesis for the pathogenesis ofrheumatoid arthritis,” Immunology Today 17:466–470 (1996); La Cava etal., “Genetic bias in immune response to a cassette shared by differentmicroorganisms in patients with rheumatoid arthritis,” J. Clin. Invest100:658–663 (1997). Each of the above reference are herein incorporatedby reference).

Materials and Methods

Preparation of DNA.

Genomic DNA is prepared from white blood cells by methods known to thoseskilled in the art utilizing ammonium acetate and isopropyl alcoholprecipitation with resuspension in TE (1 mM Tris, 0.1 mM EDTA, dH2O, pH7.5).

PCR amplification.

DNA is enzymatically amplified by the polymerase chain reaction andamplification primers that have previously been reported for DQA1 andDQB1, for level I DRB typing, for level 2 allele assignment of DRB1specificities associated with DR52, and for DR4 and DR1 (Vooter, C. E.Tissue Antigens 51:1:80–87.; Barnardo, M. C. Tissue Antigens51:3:293–300; Mitsunaga, S. Eur J Immunogenet. 25:1:15–27). Genomic DNA(5–10 μl at 80–150 μg/ml) is added to PCR mix consisting of 5 μl of 2mmole/μ1 dNTPs, 3 μl of each primer at 10 pmole/μl, 5 μl of 10× PCRbuffer, 0.5 μl of Taq polymerase (5 U/μl) and 23.5 μl sterile dH20.Total reaction volume is 50 μl. DNA is amplified for 30 cycles in a DNAThermal Cycler (Perkin Elmer Cetus) with denaturation, annealing andextension parameters that vary depending upon the locus studied. Ingeneral, annealing is at 53° C. for DQ and DRB level I, and at 60° C.for DRB 1-specific families. Positive and negative controls are includedwith each run and care is taken to avoid any source of contamination.

SSOP hybridization and detection.

After amplification, 5.0 μl samples (10% of total PCR mixture) are runon a 1.0% agarose gel to verify sufficient quantity of amplifiedproduct. The remaining portion of the samples (90% of total PCR mixture)are denatured prior to blotting and hybridization usingchem-luminescence using methods known to those skilled in the art.Membranes are washed twice with 2×SSC+0.1% SDS (0.3 M NaCl, 0.03 MNa-citrate, 0.1% SDS, dH2O, pH 7.0) for five minutes at roomtemperature, and then twice with 3 M TMAC buffer in the appropriatetemperature for the length of the probe. Fifteen-base pair probes arewashed at 50–52° C. and 18-base pair probes are washed at 59–61° C.Membranes are wetted with Lumi-Phos (Boehringer Mannheim), sealed inacetate sheets, and exposed to X-ray film for 1 to 5 minutes. Resultsare graded using 11th International Workshop criteria as follows: 1:negative (definite) 2: negative (probable) 4: indefinite 6: positive(probable) 8: positive (definite) 9: positive (definite, more thandouble intensity). If unique hybridization patterns are found in thecourse of these studies sequence analysis is done. The patient employedin the example was HLA typed by PCR.

HLA Purification.

Lymphoblastoid cell lines from RA patients are used as a source of HLAmolecules, as known to those skilled in the art. Allele andspecies-specific monoclonal antibodies, whose corresponding hybridomasare already available, are produced as ascites liquid from BALB/c miceand purified on protein A, and covalently bound to a solid support(AffiGel, Bio-Rad, Richmond, Calif.). HLA molecules are purified byimmunoaffinity chromatography from lysates of 10⁸ lymphoblastoid cells.The yield ranges from 1 to 4 mg/preparation. Purity is assessed bySDS/Page and Western blotting. Molecules in an elution buffer containing0.2% octasilglucoside are stable for more than 4 months at 4° C.

Preparation of Liposome:MHC:Antigen Complexes.

Liposomes are prepared as described in Example 1.

Flow Cytometry.

Flow cytometry is performed as described in Example 1.

Results.

T cells specific for the various combinations of the HLA, DR/DQ, and SIor dnaJp1 peptides were enumerated. T cells binding the SI or dnaJp1peptides in the context of HLA DR are more abundant (FIGS. 13A–E). Seenin the figure are PMBC from an RA patient expressing disease associatedalleles DRB* 10401 and DQ*30301 captured with DQ/PI (FIG. 13-A), DQ/SI(FIG. 13-B), DR/PI (FIG. 13-C), DR/SI (FIG. 13-D), and DQ/Mtd1 (FIG.13-E).

In order to demonstrate cross-reactivity, T cells specific for thevarious HLA/peptide combinations are sorted and RT-PCR for TCR VP geneusage is performed. The results, shown in Table III, demonstrate that Tcells specific for the self-HLA derived peptide SI cross-react with thehomologous peptide dnaJp1. Identification and isolation of T cells witha high pathogenic potential, as described in this Example 5 allowsmanipulation ex vivo to induce tolerization.

TABLE III TCR Vβ Usage of T cells Isolated by Detection with RAAssociated HLA Molecules Bound by Self or Exogenous Peptides. HLADQHLADR dnaJP1 1, 8, 12 8, 14 SI 4, 12, 21 1, 9, 14

Peripheral blood mononuclear cells were isolated from an RA patientexpressing the RA associated HLA alleles DRB*10401 and DQB*30301. Thecells were cultured for five days in the presence of IL-2 and thebacterial heat shock protein peptide dnaJP1 then sorted by flowcytometry using HLA/Antigen combinations complexed into liposomes.RT-PCR was performed on sorted cells using primers specific for theknown Vβ regions. Data are representative of two separate experiments.

The numbers in the above Table III show that of the 23 genes of the Vβgene family, cross reaction occurs with only a few even though thefamily is highly related. Additionally, the results show that individualmembers of the family can be detected using the T cell capture method ofthe invention using artificial APCs.

EXAMPLE 6 Identification, Isolation and Characterization of T CellsSpecific for Juvenile Dermatomiosytis-Related Antigens

Juvenile Dermatomiosytis is a chronic autoimmune disease of unknownetiology. It has been reported that in several patients there is anassociation between relapse of the disease and documented Streptococcuspyogenes infection (Martin, A., Ravelli, A., Albani, S., J. Peds,121:739–742 1992). Sequences shared between Streptococcus M5 protein andthe human skeletal myosin, the target of the autoimmune process havebeen reportedly identified. It has also been demonstrated that theshared sequences contain epitopes which elicit cross-reactive T cellresponses. In this Example 6, it is demonstrated that the homologoussequences are actually recognized by T cells which bear the same TCR Vβgene.

Materials and Methods

Sequence-specific oligonucleotide probe HLA typing was performed asdescribed in Example 5.

Preparation of Liposome:MHC:Antizen Complexes.

Liposome:MHC:antigen complexes are prepared as described in Example 1.

Flow Cytometry.

Flow Cytrometry is performed as in Example 1.

Two alternative methods for visualization of the antigen-specific Tcells were employed. In the first method, affinity-purified HLAmolecules were stabilized by incorporation within the interior ofliposomes using a method similar to that described in Examples 1–5 andthen incubated with biotinylated peptides which were labeled using amethod similar to that described in Examples 1–5. In a second alternatemethod, a label, in this case fluorescent (FITC), was incorporateddirectly into the liposomes themselves. Thus, the liposomes, rather thanthe peptides were labeled. FIGS. 14A–D shows data representing PBMC froma patient with JDM capture with a class I HLA and M61-1, M61-1*, M61-2,and M61-2* (FIGS. 14-A to 14-D, respectively).

Results from these experiments show that T cells which recognizehomologous peptides of either self or bacterial origin use the same TCRVβ genes, confirming cross-reactivity at a molecular level. In addition,a direct identification and enumeration of CD3+ antigen-specific T cellsis possible without detectable differences in sensitivity between thetwo labeling methods used. These results also suggest that directincorporation of a label, e.g., FITC, into the liposomes themselves, orincorporation of a labeled (e.g., biotinylated) transmembrane proteinwhich may or may not interfere with the TCR:antigen:MHC interactions,represent valid methods as alternatives in labeling of the liposome.This may be advantageous in that a label (e.g., a biotin molecule) whencomplexed with the peptide, may interfere with the interactions amongagretopes and epitopes within the TCR:antigen:MHC complex. This may beespecially true with class I MHC molecules, in which the antigen bindinggroove is open only at one end. Our diverse approaches bypass thesepotential limitations.

EXAMPLE 7 Identification and Enumeration of T Cells Specific forPeptides from Pathogenic Organisms

Physiologic, as well as vaccine-induced, immune responses to infectiousagents are based on T cell recognition of, and reactivity to,immunogenic epitopes of the infectious agents. In several instances,immunogenic epitopes of microorganisms employed in vaccines, and theirHLA restrictions, have been identified. One example is the influenzavaccine.

In this Example, quantitative and qualitative T cell responseabnormalities in response to influenza vaccination can be identified.Peptides encompassing the major epitopes of the influenza virus areemployed for analysis. Antigen specific T cells are identified andisolated as described in Example 1. The population to be screened iscomprised of elderly persons who are vaccinated and then screened forimmune responses. Antigen specific T cells are quantified and isolatedfrom both the responder and non-responder groups. Phenotypical andfunctional characteristics are then evaluated, and the results comparedbetween the two groups to analyze whether hyporesponsiveness in some ofthe vaccinated subjects is related to lack of specific T cells.

PBMC are incubated with the relevant and control peptides for five daysin order to increase precursor frequency. Cells are then incubated withHLA-peptide complexes, and positive cells are enumerated by FACS.Commercially available cell lines are the source of soluble HLAmolecules (Corriel Cell Repository).

Antigen specific T cells are evaluated for the production of cytokineswhich are related to either effective responses to viruses (IFN, IL-2)or associated with anergy (as in the case of using, for example, IL-10).This analysis elucidates differences, if any, between antigen specific Tcells of responders versus non-responders. Hence, important functionaldifferences are evaluated at an antigen-specific T cell level. Membranephenotype of the isolated antigen specific T cells is evaluated, withparticular attention to early activation markers, such as CD69, andmemory, such as CD45RO. This test helps distinguishing anamnestic fromrecently induced responses. mRNA is purified and RT-PCR using Vβ chainfor the T cell receptor specific primers is performed. The definition ofthe T cell receptor (TCR) used by the various subjects may help inpointing out differences between responders and non-responders due togenetic differences in the TCR repertoire. This shows the potential forthe technology of the current invention to discriminate betweenanamnestic and recall immune responses at the level of antigen-specificT cells.

Materials and Methods

Lymphocyte proliferation assays.

Preliminary experiments are performed to identify the optimal conditionsfor culture. Initially, cell cultures are performed in duplicate forthree to seven days, using 5 million cells/well. (All antigens haveshown a range of optimal responses between 1 and 10 μg/ml). Theproliferation assays are conducted as controls to compare against the Tcell capture specificity of the invention.

Lymphocyte cytotoxicity tests.

Cytotoxic responses are measured using a LDH-release kit (Promega). Thetests are performed according to manufacturer's instructions. Effectorsare incubated for 5 days with IL2 and the relevant peptides or withirrelevant antigens. Targets are irradiated autologous PBMC or, whenavailable, EBV-transformed lymphoblastoid cell lines. Targets are pulsedwith the relevant antigens overnight. The cytotoxicity assays areconducted as controls to compare against the T cell capture specificityof the invention.

Antigens.

Synthetic peptides (Matrix 58–66:GILGFVFTL (Seq. Id. No. 5);Nucleoprotein 82–94:VKLGEFYNQ (Seq. Id. No. 6)) are purchased fromResearch Genetics.

MHC purification.

Commercially available lymphoblastoid cell lines (Corriel CellRepository) are used as a source of MHC molecules and purified byimmunoaffinity chromatography using anti-HLA class I antibodies in amanner similar to that shown in Example 5.

Liposome:MHC:antigen complexes are prepared as described in Example 1.Antigen specific T cells are prepared as described in Example 1.Intracellular immunofluorescence staining of cytokines was performed asdescribed in Example 10.

Cytokine measurement by RT/PCR.

mRNA is extracted from approximately 7×10⁶ cells by using OligotexDirect mRNA Kit (Qiagen, Chatsworth, Calif.). mRNA isreverse-transcribed into cDNA with the oligo dT primer (RT-PCR Kit,Stratagene, La Jolla, Calif.). Two μl of single strand cDNA areamplified using the cytokine specific forward and reverse primer sets,IL-2, IL4, TNF-a, INF-γ. Quantitative measurement of various cytokinesusing competitive PCR is performed (Biosource reagents). In this method,a known copy number of an exogenously synthesized DNA used as aninternal control sequence (ICS) is mixed with the sample prior toamplification. The ICS is constructed to contain identical primerbinding sites as the cytokine to be analyzed, and a unique binding sitethat allows the resulting amplicon to be distinguished from the cytokineproduct. Detection of the amplification product is by non-radioactivemicroplate techniques.

T cell receptor analysis by PCR.

Messenger RNA is extracted from a minimum of 70 cells by usingMicro-FastTrack Kit (Invitrogen, Carlsbad, Calif.). The yield of mRNA isabout 2 μg that is resuspended into 20 μl of water. Two μl of mRNA foreach reaction is reverse-transcribed into single strand cDNA with theoligo dT primer (cDNA Cycle Kit, Invitrogen, Carlsbad, Calif.). 1.5 μlof single strand cDNAs are amplified with constant primer and various Vregion primers (Vb1–Vb24), the sequences of which may be designed bythose knowledgeable in the art. For example, 20 pico moles of eachprimer and 1.25 units of Taq polymerase (Boehringer Mannheim, Germany)are used. The total volume is 25 μl. The cycling parameters for PCR isindicated as: heat PCR reaction mixture without Taq and dNTPs at 94° C.for 4 min, then perform 40 cycles of 30 seconds at 94° C., 30 seconds at58° C. and 30 seconds at 72° C. The final elongation is 7 minutes at 72°C. Add 0.5 μl of 100 mM dNTPs and 0.25 μl of Taq Polymerase (1.25 units,Boehringer Mannheim) at end of 58° C. of first cycle. The PCR-amplifiedproducts are analyzed on 4% agarose gel.

In summary, the experiments described in this Example 7 are accomplishedaccording to the following steps:

-   -   i) MHC typing by PCR of the test sample derived from a test        subject, (e.g. a patient that is to receive treatment against an        infectious agent);    -   ii) Purification by immunoaffinity chromatography or by other        methods known to those skilled in the art, of MHC molecules        corresponding to that of the test subject's. The source for        these molecules may be directly from an individual or from cell        lines homozygous for the HLA alleles.    -   iii) Synthesis of liposomes containing MHC:peptide complexes.        (These liposomes may be tagged according to any of the methods        described herein or known by those in the art.);    -   iv) Incubation of PBMC from the test subject with the        MHC:peptide tagged liposomes, and;    -   v) counting of the binding T cells for the purpose of        determining which peptides have bound thereby indicating the        specific peptides effective in binding antigen-specific T cells.

Methods described in this Example are useful in determining the numberof vaccine-specific T cells in peripheral blood, both in normal andimmune compromised individuals, for example, as a tool for decisionsregarding the opportunity for vaccination in immune compromisedsubjects, and in determining the efficacy of vaccination, measured asincrease in the number of vaccine-specific T cells after vaccination.

EXAMPLE 8 Diagnosis and Immunomodulation of Allergic Disease

Allergy is mediated by release of pro-inflammatory and vasoactivemediators triggered by binding of allergen specific immunoglobulins withreceptors of inflammatory cells at the site of allergen contact withcellular tissues. The production of allergen specificimmunoglobulins-appears to be mediated by strong interactions between Band T cells, an example being Casein in the pathogenesis of lactoseintolerance (Albani, S. Annals of Allergy. 63:12:489–492.). It istherefore of importance to have the possibility to identify allergenspecific T cells, isolate them and manipulate them ex vivo. An outlineof the strategy to be employed is:

-   -   i) MHC typing of the test subject by PCR technology;    -   ii) Identification of candidate “allergenic” peptides, based on        current knowledge of the field. (When a candidate epitope on a        given proteic allergen will not be already available,        computerized analysis of MHC binding motifs will enable the        identification of candidate peptides to be used.);    -   iii) Synthesis of liposomes containing relevant MHC:peptide        complexes. (Such liposomes can be tagged using any of the        techniques described herein or known by those in the art.);    -   iv) Incubation of PBMC of the test subject with the        MHC:peptide:tagged liposomes complexes;    -   v) Identification and enumeration of allergen specific T cells;    -   vi) manipulation ex vivo of such cells, by stimulation in        culture with the antigenic peptide in the presence of stimuli        related to induction of Th-1 phenotype.

Materials and Methods

Lymphocyte proliferation assays are performed as described in Example 7.

Lymphocyte cytotoxicity tests are performed as described in Example 7.

Antigens.

Peptides are identified based on scanning the sequences of the proposedallergens for MHC-binding motifs and synthesized according to standardpeptide synthesis methods know to those in the art.

MHC purification is performed as described in Example 7.

Liposome MHC:antigen complexes are prepared as described in Example 1.

T cells, generated as described in Example 1, are captured by separationusing flow cytometry as described in Example 1.

Intracellular immunofluorescence staining of cytokines, is performed asdescribed in Example 5.

Cytokine measurement by RT/PCR is performed as described in Example 7.

T cell receptor analysis by PCR is performed as described in Example 7.

This Example therefore shows that artificial APCs may be used todiagnose and monitor progress of therapies and modulation level ofspecific responses in a patient's T cells.

EXAMPLE 9 Identification of Cancer-Specific T Cell Epitopes

It is commonly accepted that lack of immunity to cancer (e.g.,melanoma), depends on low antigenicity of the neoplasm, as well as onfunctional and/or numerical deficiencies of antigen-specific T cells.

Several therapeutical approaches are currently underway to improve theefficiency of recognition of and reactivity to cancer antigens by Tcells. Unfortunately, the efficiency of the treatment can be measured,to date, only in terms of clinical outcome. This Example 9 describes asolution to this problem, by enabling identification, enumeration,characterization and possible manipulation of cancer-specific T cells.Hence, efficiency of the treatment will be measured in terms of increasein antigen-specific precursor frequency, and also in terms of functionaloutcome of antigen recognition. This latter aspect is of particularimportance in those instances where the therapy is aimed at activationof otherwise dormant T cells.

The following protocol is applied:

-   -   i) MHC typing by PCR of the test subject;    -   ii) Identification of candidate peptides. For example, sources        for antigen in the treatment of melanoma include MAGE-1, MAGE-3,        MART-1/melan-A, gp100, tyrosinase, gp75, gp15, CDK4 and        beta-catenin. When a candidate epitope on a given protein        allergen will not be already available, computerized analysis of        MHC binding motifs will enable the identification of candidate        peptides to be used;    -   iii) Synthesis of liposomes containing relevant MHC:peptide        complexes;    -   iv) Incubation of PBMC of the test subject with the        MHC:peptide:tagged liposomes complexes;    -   v) Identification and enumeration of antigen specific T cells.

Materials and Methods

Lymphocyte proliferation assays are performed as described in Example 7.

Lymphocyte cytotoxicity tests are performed as described in Example 7.

Antigens.

Synthetic peptides will be identified based on scanning the sequences ofthe proposed allergens for HLA-binding motifs.

MHC purification.

MHC purification is performed as described in Example 7. Commerciallyavailable lymphoblastoid cell lines (Corriel Cell Repository) are usedas a source of MHC molecules, and purified by immunoaffinitychromatography using anti-MHC class I antibodies, available in ourlaboratory.

Liposome MHC:antigen complexes are prepared as described in Example 1.

T cells, generated as described in Example 1, are captured by separationusing flow cytometry as described in Example 1.

Intracellular immunofluorescence staining of cytokines, is performed asdescribed in Example 5.

Cytokine measurement by RT/PCR is performed as described in Example 7.

T cell receptor analysis by PCR is performed as described in Example 7.

As shown, a peptide based method for monitoring cancer therapy isestablished using antigen-specific artificial APCs. This methods allowsfor studying the progress of the therapy and the state of the cancer.

EXAMPLE 10 Methods for Distinguishing “Bystander T Cells” fromAntigen-Specific T Cells

The experiments described in this Example demonstrate thatantigen-specific T cells can be identified and enumerated anddistinguished from antigen non-specific T cells present in the sametissue. These latter cells (so-called “bystander T cells”) mayparticipate in pathogenic processes. This has particular relevance forautoimmune and allergic diseases, where the initiating event may be therecognition of a pathogenic antigen by a specific T cell population.This interaction leads to production of pro-inflammatory or pro-allergicmediators (i.e., cytokines) by the antigen-specific T cells. Thecytokine cascade will subsequently involve T cells that are not specificfor the antigen (bystander T cells), which may then participate in thepathogenic processes, amplifying significantly the degree of the damage.It is important in a clinical or research setting to discriminatebetween the antigen-specific and bystander populations in order toevaluate, for instance, the efficacy of a treatment. The strategy may besummarized as follows;

-   -   i) Identification of candidate peptides, based on current        knowledge of the field. When a candidate epitope on a given        protein antigen is not already available, computerized analysis        of MHC binding motifs will enable the identification of        candidate peptides to be used in the “T cell capture”. Such        analysis is standard practice to those skilled in the art;    -   ii) Synthesis of liposomes containing relevant MHC:peptide        complexes. Such liposomes can be tagged using any of the        techniques described herein;    -   iii) Incubation of PBMC of the test subject with the        MHC:peptide:tagged liposomes complexes;    -   iv) Identification and enumeration of antigen specific T cells.

Materials and Methods

Lymphocyte proliferation assays.

Preliminary experiments may be performed to identify the optimalconditions for culture. Cell cultures may be performed in duplicate forthree to seven days, at 5 million cells/well. All antigens have shown arange of optimal responses between 1 and 10 μg/ml.

Lymphocyte cytotoxicity tests.

Cytotoxic responses may be measured using a LDH-release kit(commercially available). The tests may be performed according tomanufacturer's instructions. Effectors may be incubated for 5 days withIL2 and either the relevant peptides or with irrelevant peptideantigens. Targets may be irradiated autologous PBMC or, when available,EBV-transformed lymphoblastoid cell lines. Targets may be pulsed withthe relevant antigens overnight.

Antigens.

Synthetic peptides are identified based on scanning the sequences of theproposed allergens for MHC-binding motifs.

MHC purification.

Commercially available lymphoblastoid cell lines (Corriel CellRepository) may be used as a source of MHC molecules and purified byimmunoaffinity chromatography using anti-MHC class I antibodies.

Preparation of Liposome MHC:Ag Complexes.

Preparation of APCs are the same as that performed for Example 1.

FACS. A Beckton Dickenson FACS Star with LYSIS II software was used tovisualize cells. Sortings were performed by the Flow Cytometry Core atUCSD.

T cell Capture.

T cells, generated as described, are captured by separation using flowcytometry. Bulk sorted cells are either cultured as described,immediately used for DNA analysis, or used directly for reanalysis usingFACS. Yields from bulk sortings range from 2000–16,000. Single cellsorts utilized 96 well culture plates containing media previouslydescribed. Fresh irradiated APCs are added to the single cell culturesonce per week, at which time analysis of clonal expansion is performed.Of 96 wells of sorted “events”, generally 8–12 show good expansion overa six week period.

Intracellular immunofluorescence staining of cytokines.

Human PBMC are isolated by density centrifugation and stimulated for 36hours with peptides at 10 μg/ml in the presence of 2 μM monensin. Cellsare washed in PBS with 2% FCS and incubated with Fc-block for 5 min. at4° C. PE-conjugated anti-CD3 monoclonal antibodies (mABs) are added andcells are incubated for 30 min. at 4° C. Cells are washed, fixed 20 min.at 4° C., and resuspended in a solution containing either FITCconjugated anti-IFNγ mAbs or FITC conjugated anti-IL4 or IL2 mAbs. Cellsare incubated for 30 min at 4° C., washed twice in PBS with 2% FCS andtheir fluorescence measured using a Becton Dickinson FACScan. Flow dataare analyzed using Lysis II software (Becton Dickinson).

Cytokine measurement by RT/PCR.

Messenger RNA is extracted from approximately 7×10⁶ cells by usingOligotex Direct mRNA Kit (Qiagen, Chatsworth, Calif.). The mRNA isreverse-transcribed into cDNA using oligo dT primer (RT-PCR Kit,Stratagene, La Jolla, Calif.). Two μl of single strand cDNA areamplified using the cytokine specific forward and reverse primer setsfor IL-2, IL4, TNF-α, and INF-γ. Quantitative measurement of the variouscytokines was carried out using competitive PCR (Biosource). In thismethod, a known copy number of an exogenously synthesized DNA (ICS) ismixed with the sample prior to amplification. The ICS has beenconstructed to contain identical primer binding sites as the cytokine tobe analyzed, and a unique binding site that allows the resultingamplicon to be distinguished from the cytokine product. Detection ofamplicons is allowed by non-radioactive microplate techniques.

T cell receptor analysis by PCR.

Messenger RNA is extracted from a minimum of 70 cells by usingMicro-FastTrack Kit (Invitrogen, Carlsbad, Calif.). The yield of mRNA isabout 2 μg that is resuspended into 20 μl of water. 2 μl of mRNA foreach reaction is reverse-transcribed into single strand cDNA with theoligo dT primer (cDNA Cycle Kit, Invitrogen, Carlsbad, Calif.). 1.5 μlof single strand cDNAs are amplified with constant primer and differentV region primers (e.g., Vb1–Vb24). 20 pico moles of each primer and 1.25units of Taq polymerase (Boehringer Mannheim, Germany) were used. Thetotal volume is 25 μl. The cycling parameters for PCR are: heat PCRreaction mixture without Taq and dNTPs at 94° C. for 4 min, performanceof 40 cycles for 30 seconds at 94° C., 30 seconds at 58° C. and 30seconds at 72° C. The final elongation is 7 minutes at 72° C. Add 0.5 μlof 100 mM dNTPs and 0.25 μl of Taq Polymerase (1.25 units, BoehringerMannheim) at end of 58° C. of first cycle. The PCR-amplified productswere analyzed on 4% agarose gel. The sequences of Vβ-specific primerswhich may be used may be designed by those knowledgeable in the art.

This example therefore shows that antigen-specific immunotherapy can beused to influence populations of T cells having different specificitywhich participate in the pathogenic process.

EXAMPLE 11 Immunoaffinity Chromatography for Positive Selection ofAntigen-Specific T Cells

As demonstrated in this Example, MHC:antigen complexes may beincorporated into liposomes as described in certain embodiments of theinvention above and bound to a solid support. By orienting the complexesso as to optimize the chance of interaction between the T cell receptorand the complexes, antigen-specific T cells may be isolated frombiological samples.

Immunoaffinity chromatography columns.

MHC:peptide complexes may be bound to hydrazide coated glass beads.Alternatively, molecules having affinity for irrelevant molecules may bebound to glass beads coated with a hydrazide linker and may be used tobind a irrelevant molecule-containing liposome:MHC:peptide complexes. Aslurry of either of the above coated bead solid supports is incorporatedinto a compartment of a column such as that shown in FIG. 8 in acontrolled fashion. Polymorphic T cell populations contained in a liquidmedium are then introduced into the column, and the mixture is incubatedat room temperature for 30 minutes, with gentle turning of the column. Tcells with irrelevant specificity are not bound by the slurry and willflow through. Peptide or antigen-specific T cells that bind duringmixing are then removed from the solid supports by incubating the columnat 4° C. for 30 minutes.

EXAMPLE 12 Ex Vivo Depletion of Cells Related to a Pathogenic Process:Antigen-Specific Leukapheresis

In certain situations, it is desirable to deplete living systems of Tcells having specificity for a given antigen. In the case ofautoimmunity or allergy, these T cells may be involved in pathogenicprocesses and their depletion may therefore induce clinical improvement.Likewise, in the case of transplantation, recognition of a donor'sepitopes and ideotopes as non-self will cause graft vs. host rejectionof a transplanted organ. In such instances, depletion of the recipient'sT cell population that recognize the foreign epitopes/ideotopes isbeneficial. Depletion of such reactive T cells may be accomplished byconnecting in line with the general circulation of a patient a devicecomprising MHC:antigen complexes bound to a solid support and orientedto optimize TCR:complex interaction. For example, MHC:peptide complexesmay be associated in liposomes containing irrelevant molecules which maythemselves be captured by glass beads coated with molecules havingspecificity for the irrelevant molecules. Appropriate filters,sterilization and heating procedures may be used in a manner similar tothat currently employed in conventional leukaphoresis procedures. Inoperation, whole blood or blood enriched for T cells is allowed to flowthrough the device. Antigen-specific T cells are bound and eliminatedfrom solution. The slurry is continuously stirred, and after appropriatetime periods portions of it are incubated at 4° C. in order to elute theantigen-specific T cells that have bound to the solid support associatedMHC:antigen complexes. For clinical conditions where suchantigen-specific T cells are harmful, such cells can be simplydiscarded.

The specific procedure with respect to autoimmunity comprises:

(i) Preparing a column in which the MHC:antigen:accessory molecule:othermolecule complex is designed to interact with T cells which may beinvolved in the pathogenesis of autoimmune disease using methodsdescribed in Example 11.

(ii) Allowing blood from the patient to flow through the columncontaining artificial APC prepared in step (i), by which means antigenspecific T cells will be retained.

The specific procedure with respect to transplantation comprises:

(i) Preparing a column in which the MHC:antigen:accessory molecule:othermolecule complex is designed to interact with T cells which may beinvolved in either the graft vs. host disease of bone marrow transplantor the pathogenesis of graft destruction in allogenic solid organtransplant using methods described in Example 11.

(ii) Allowing blood from the patient to flow through the columncontaining artificial APC prepared in step (i), by which means antigenspecific T cells will be retained.

EXAMPLE 13 Ex Vivo Manipulation of Antigen Specific T Cells:Bidirectional Switch From Th1 to Th2-Type Functional Phenotype

In the case of infectious disease or cancer, it may be beneficial toisolate cells with a given antigen specificity in order to change theirfunctional phenotype, for example, by manipulating such cells using theartificial antigen presenting cells described in this Example.Manipulated cells can then be reintroduced for immunomoduation.

Antigen-specific T cells, isolated according to Example 1 or Example 2,are incubated with the appropriate variant of the artificial antigenpresenting cells according to the following examples.

Expansion of antigen-specific T cells.

Isolated cells (10,000/ml) are incubated in standard culture medium withAPC expressing the relevant MHC:peptide combination, ICAMI as adhesionmolecule to facilitate initial interaction, and anti-CD28 transmembraneantibody. The latter is employed as a co-stimulatory molecule to induceT cell proliferation without affecting Th bias. This type of approach isthe antigen-specific equivalent of the recent approach at T cellexpansion using anti-CD3/anti-CD28 molecules. Up to 20 replicativecycles have been obtained in this system, which represents a validalternative to T cell expansion and cloning using autologous APCsystems, often an insurmountable hurdle in human systems.

Expansion and immunomodulation of cells from a Th1 to a Th2 functionalphenotype.

i) In one expansion, B7-1 may be used as a co-stimulatory moleculeinstead of anti-CD28. The cells obtained from this treatment may exertan immunoregulatory function in the autoimmunity condition.

ii) In another expansion, B7-2 may be used as a co-stimulatory moleculeinstead of anti-Cd28. Cells obtained from this treatment may haveimmunomodulatory properties in settings such as cancer or infectiousdisease.

Specifically, an artificial APC comprises MHC:peptide complexesstabilized into liposomes, with the addition of transmembrane proteinswhich accomplish co-stimulatory functions. The molecules may compriseany or all of the following:

-   -   a) ICAMI as adhesion molecule to facilitate initial interaction        between the T cell and the APC.    -   b) anti-CD28 transmembrane antibody is employed as a        co-stimulatory molecule which may induce T cell proliferation        without affecting Th bias. This type of approach is the        antigen-specific equivalent of T cell expansion using        anti-CD3/anti-CD28 molecules. Up to 20 replicative cycles have        been obtained in this system, which represents a valid        alternative to T cell expansion and cloning using autologous APC        systems.    -   c) B7.1 may be used instead of anti-CD28. The cells obtained        from this treatment may exert immunoregulatory function in        autoimmunity.    -   d) B7.2 may be used instead of anti-CD28. The cells obtained        from this treatment may have immunomodulatory properties in        settings such as cancer or infectious disease.

All of the aforementioned molecules are transmembrane proteins which canbe incorporated into liposomes according to above examples, or as analternative, bound to a solid support as in above examples.

Isolation and immunomodulation of antigen specific T cells.

Antigen-specific T cells isolated according to the above examples areincubated with the appropriate variant of the APC according to thedesired objective, i.e. expansion, functional phenotype switch, etc.

EXAMPLE 14 Monitoring Immunological Outcome of Intervention ofAntigen-Specific T Cells by Correlation of Clinical Outcome with T CellPhenotype

In this example, the invention is applied to evaluating the clinicaloutcome of treatment regimens by correlating the phenotype ofantigen-specific T cells with clinical outcome. Specifically, theinvention is applicable to clinical monitoring and clinical trials wherethere is a need to evaluate the effectiveness of artificial APC inducedT cell responses. The antigen-specific T cells associated with aresponse (or disease state) are identified followed by theidentification of their functional phenotype. The phenotype identifiedis then correlated and monitored against the progression of a patient'sresponse to various treatment regimens.

EXAMPLE 15 Interaction of Artificial APCs with T Cells to Induce Cappingof Transmembrane Proteins

The immune synapse is the cluster of transmembrane molecules whichensures specific interaction between antigen specific T cells andantigen presenting cells. The outcome of these interactions is antigenspecific response by the T cells, mediated by signaling through the TCR.Several factors contribute to significant quantitative and qualitativedifferences in the response provided by the T cell. These factorsinclude affinity of interaction between MHC and peptide, and between theTCR and the MHC/peptide complex, the number of moieties Available forinteraction, and the relative concentration of the moieties availablefor interaction. In a physiological situation, the triggering number ofinteracting molecules is achieved by “capping”, a phenomenon, whichoccurs when transmembrane molecules are allowed to freely migrate todefinite zones of the membrane, usually upon initial interaction betweentwo of the ligands involved, in this case the TCR and the MHC/peptidecomplex. In order to emulate such physiologic mechanisms, we employedartificial APCs loaded with the combination IA^(d)/OVA, and hybridoma8DO as the specific T cells. To visualize free movement of the TCR inthe T cell membrane, we employed a system where FITC-conjugated choleratoxin, a molecule known to combine with the intracellular portion oftransmembrane molecules, is introduced into the T cells. We show herefor the first time that artificial APCs can effectively emulate thephysiologic interactions between T cells and APC, particularly withrespect to allowing migration of molecules whose proper density is anessential requirement to induce T cell activation. Our system has alsothe potential to be a tool to study physiological mechanisms of T cellactivation, and to manipulate the intensity and quality of T cellresponse. This could be accomplished by controlling the affinity of theinteraction, and by adding the proper co-stimulatory and adhesionmolecules to the artificial APC.

Approximately 100 MHC/peptide binding sites are available on eachartificial APC for interaction with a single T cell, increasingtherefore the likelihood that artificial APCs will engage, throughmultiple interactions, low-affinity T cells. This may provide asignificant advantage over current methods for the identification of lowaffinity, class II restricted antigen-specific T cells, such as the onesoften involved in physiologic regulatory mechanisms or in diseaserelated autoimmune responses.

We first identified the optimal incubation times for the artificial APCwith the T cells, (i.e., about 20 minutes) at different stages ofevolution in the capping mechanism. Unlike systems where the interactingmolecules are fixed on planar membranes, interactions between T cellsand artificial APCs occur in culture medium, and rely on random movementfor molecular interactions. This results in the optimal period for aresult to be based on formation of “capping” of the TCR in the cellmembrane rather than just binding of the TCR and target moieties. In theexperiments shown in FIGS. 16–18, we incubated the artificial APC with8DO cells for 20 minutes, and evaluated the capping by confocalmicroscopy. In FIGS. 16A–D, we visualized co-migration of the choleraraft and the TCR, by incubating T cells with PE-conjugated monoclonalantibody (Pharmigen). TCR molecules are uniformly distributed in thecells membrane, as expected for cells, which are not interacting withstimulatory triggers. In experiments described in FIG. 17A–D, wevisualized interaction of TCR by using the cholera toxin raft, and theMHC in the artificial APC membrane using a specific anti-MHC (Pharmigen)monoclonal, Alexa-red conjugated. In the field shown, the phenomenon ofprogressive migration of the TCR molecules toward the point of initialinteraction of the artificial APC with the TCR is evident at differentstages for different cells. In experiments described in FIGS. 18A–D, wevisualized the TCR using an Alexa red-conjugated anti CD3 monoclonal(Molecular Probes, Eugene, Oreg.) and the liposomes usingFITC-conjugated streptavidin which bound to the biotin at the N-terminalof the OVA peptide. The results of this visualization procedure in FIG.18 are overlapping with those shown in FIG. 17. Hence, we show here forthe first time that artificial APCs can effectively emulate thephysiologic interactions between T cells and APCs, particularly withrespect to allowing migration of molecules whose proper density is anessential requirement to induce T cell activation. Our system alsoprovides for studying physiological mechanisms of T cell activation andthe manipulation of the intensity and quality of T cell response. This“modulation” of T cell response could further be accomplished bycontrolling the affinity of the interaction by adding the properco-stimulatory and adhesion molecules to the artificial APC. Anadditional preferred advantage of the system of the invention is that itallows free movement of transmembrane proteins, enabling the sequentialorder of interactions among ligands, which occur upon first contact ofthe TCR with the APC in the immune system.

-   -   Immunohistochemistry Methods for FIGS. 16–18

Liposomes and 0.5×10⁵ cells were cytospinned at 300 g for 5 min onpoly-L-lysine coated slides (Sigma, St. Louis, Mo.). The cells werefixed for 10 minutes in 4% Paraformaldehyde, washed in PBS and thenincubated for 1 hours with the antibodies anti-CD3 1Ad (Pharmingen, SanDiego, Calif.), at a concentration of 2–10 μg/ml in I-Block blockingbuffer (Tropix, Bedford, Mass.). Alexa 568 (red) species-specificsecondary antibodies (Molecular Probes, Oreg.) were used to visualizethe proteins. Coverslips were washed successively in PBS and deionizedH₂O for 5 min and mounted in Flouromount (Fisher, Calif.). Images wereobtained with a Zeiss Confocal Laser Microscope.

EXAMPLE 16 Identification of Class II Restricted T Cells in PolymorphicPopulations

Identification of antigen-specific, class II restricted T cells is muchmore difficult than the same identification analysis for class Irestricted T cells. These difficulties stem from the lower affinity ofinteraction of TCR/peptide/MHC for class II restricted responses, whencompared to class I. Known methodologies have identified only highaffinity antigen specific T cells based on soluble recombinant moleculesthat provide anywhere from one to four interaction sites for TCRbinding, wherein steric hindrance problems are thought to affect correctfolding of the recombinant molecules and their interactions with theTCR. In contrast, our results show that MHC/biotinylated peptidecomplexes can effectively visualize class II-restricted hybridoma Tcells regardless of affinity.

To evaluate the efficiency of our artificial APC system to identifyClass II restricted antigen specific T cells, we immunized BALB/c micewith the peptide Iα52. Iα52, is a naturally processed, abundantlypresented IE^(d)-derived peptide, which has previously been described asantigenic in BALB/c mice. Cells from regional draining lymphnodes, bothfrom IFA only and Iα52 immunized mice, were harvested. These cells werethen incubated with artificial APC presenting IA^(d)/Iα52 complexes. Asshown in FIG. 25B, 5.4% of CD4+ T cells were Iα52 specific, while incomparison, only 0.7% CD4 cells were specific for the peptide in theIFA-only immunized mice (FIG. 25A). These data related well toantigen-specific T cell proliferation, measured by standard thymidineincorporation assay (FIG. 26).

Capture of T cells by Artificial APCs is Effective in IdentifyingPolyclonal Class II Restricted Human T Cells

To identify human antigen-specific T cells, we employed as modelantigens a system comprising Pan-DR binder peptides (PADRE) ofcomparable affinity, and the influenza hemoagglutinin HA peptide. Thepeptides used were pan-DR binding peptide 965.10 PADRE (K(X)VAAWTLKAASeq. Id. No. 7), HA 307–319 (PKYVKQNTLKLAT Seq. Id. No. 8), and 1Adbinding peptide OVA 323–339 (ISQAVHAAHAEINEAGR Seq. Id. No. 9). Peptideswere synthesized as C-terminal amides, purified by reversed-phase HPLC,and checked by fast atom bombardment mass spectrometry. For use inMHC-binding assays and the T cell capture, peptides were biotinylatedduring peptide synthesis (only one biotin molecule, at the n-terminus,100% biotinylation).

The choice of such antigens was based on the concept that a high numberof polyclonal T cells could be found in PBMC if a peptide with highaffinity would be employed in the assay. We first defined the optimalmolar concentration to employ in loading PADRE pan-DR peptideKXVAAWTLKAA Seq. Id. No. 7, and showed the specificity of interactionbetween the PADRE peptide and the HLA molecules. T cells from a normalHLA DRB 1*401 donor were first tested for the number of PADRE andHA-specific T cells (FIG. 23A), and then cultured with 10 μg/ml of PADREfor ten days. As shown in FIG. 23B, PADRE-specific T cells were expandedto 8.1%, while the number of cells specific for the control HA peptideactually decreased from 1.0 to 0.3% (FIG. 23D). Interaction betweenantigen-specific T cells and artificial APC was, as expected, dependingon availability of TCR and HLA/peptide molecules. Our experiment furthershowed that binding was inhibited by addition of anti-HLA antibody priorto incubation of artificial APCs with the T cells. Moreover, both totalcell number and MFI were reduced by 62% when compared with panel 23Bupon competition between biotinylated and non-biotinylated PADREpeptides. This control further supports the specificity of theinteractions between the various components of the system, and rules outthe possibility of steric hindrance from the biotin at the n-terminal ofthe peptide in interfering with the necessary molecular interactions.

T cell capture by artificial APCs is more sensitive than measurement ofcytokine production to assess the number of antigen specific T cells ina culture.

Measurement of production of cytokines in response to an antigen isoften used to estimate the number of antigen specific T cells. Tocompare sensitivity of measuring cytokine production against T cellcapture by artificial APCs, we harvested culture supernatants fromPADRE-stimulated cultures, at the fourth and sixteenth day of cultureand measured concentrations of IL-2 and IFN γ by sandwich ELISA. Asshown in FIG. 24, the differences in IL-2 and IFN-γ production betweenthe two time points were not as evident as the increase inPADRE-specific T cells, as measured by our T cell capture method (FIGS.23A–F). Hence, from the comparisons of the T cell capture usingartificial APCs with antigen induced T cell proliferation (FIGS. 25 and26) and cytokine production (FIG. 24) it appears evident that T cellcapture using artificial APCs is a tool for sensitive measurement ofantigen-specific responses. The advantage of identifying specific Tcells by T cell capture is also important for the fact that T cells maynot directly participate in antigenic responses while retaining antigenspecificity. This phenomenon may also provide an important tool for theunderstanding of the regulation of T cell responses to antigens.

Methods

Short Term T Cell Line

Peripheral Blood Mononuclear Cells (PBMC) from healthy donors werestimulated in vitro with the PADRE peptide. PBMC were isolated using aFicoll-Isopaque gradient and stimulated in vitro with 10 μg/ml PADREpeptide in a 24 well tissue culture plate (Costar, Cambridge, Ma) at4×10⁶ cells per well. The cells were cultured in RPMI containing 15% ABserum, at 37° C. and 5% CO₂. At days 4 and 7, media were replaced withfresh medium containing recombinant human Interleukin-2 (IL-2) at afinal concentration of 10 ng/ml. Culture supernatants taken at day 4were analyzed for production of IL-2 and Interferon-γ by ELISA. At day10 viable cells were harvested and analyzed in a T cell capture asdescribed below.

Animals

Balb/c mice were obtained from Harlan. Mice were 4–6 weeks old at thebeginning of each experiment.

Immunizations

Balb/c mice were immunized with 100 mg of OVA 323–339 in CompleteFreundis Adjuvant (CFA) subcutaneous, followed by immunizations with 100mg of OVA 323–339 in Incomplete Freundis Adjuvant (IFA) at days 7, 14and 21 after the initial immunization. A subgroup of mice was sacrificed5 days after each immunization.

Preparation of MHC

Balb/c MHC class II molecules I-Ad and I-Ed were purified from thelysate of B cell lymphoma A20.11 by immunoanaffinity chromatographyusing anti-IAd monoclonal antibody MKD6 (Pierce) and anti-IEk monoclonalantibody 14-4-4S, essentially as previously described.

Human MHC Class II DRB1*0401 and MHC Class I molecules were purifiedfrom the lysate of the Epstein Barr virus transformed B cell lines.

Affinity purified MHC molecules were solubilized in a TRIS buffercontaining 50 mM diethylamine and 0.2% n-acetyl-octyl-glucopyranoside(Calbiochem, San Diego). Conditions tested: pH5 and 7, RT and 370° C.,16–24–40 hr peptide loading.

Competition/inhibition of non-biotinylated PADRE peptide on binding toHLA DR4*0401.

A modified ELSA using soluble HLA-DR 0401 assessed thecompetition/inhibition of biotinylated and non-biotinylated PADREpeptide. A ten-fold molar excess of biotinylated PADRE peptide wasincubated with affinity purified DR4*0401 at pH 7.0 for 16 hrs in roomtemperature. In addition, a 250 fold molar excess of non-biotinylatedPADRE was added. After 16 hours the complexes were transferred to a96-well flat bottom ELISA plate (Costar) coated with an anti-DR captureantibody. The excess of unbound peptides and complexes were removedtrough extensive washing with buffer. A 1:20,000 solution ofNeutravidin-HRP was then added to the wells and incubated for 40 minutesin room temperature. After washing, a TMB developing solution was addedand developed for approximately 15 minutes. 1M H3PO4 was used as a stopsolution. The optical density was read at 450–650 nm using a micro-platereader. Delta Soft program was used to analyze the data.

Preparation of Cells

Cells were washed twice in staining buffer (PBS with 2% PCS and 0.05%sodium azide) and then incubated at 4° C. for 20 minutes in blockingbuffer (staining buffer with 10% FCS, for mouse cells FcBlock(Pharmingen) is used). The cells were stained for the surface markersand isotype controls for 20 minutes at 4° C., and washed twice andresuspended in staining buffer. The following monoclonal antibodies wereused: PE-, CY- and FITC-labeled anti-mouse and anti-human CD3 and CD4.

T Cell Capture

The cells were incubated with the MHC-peptide liposomes for 20 minutesat room temperature. Before acquisition on the FACScan, cells werewashed twice (5 minutes, 500 g) and resuspended in staining buffer.Partial inhibition of the interaction of cells to the liposomes wasachieved with monoclonal antibodies against T cell receptor or MHC. Forinhibition through partial blocking of the T cell receptor, cells wereincubated for 20 minutes with 50 μg/ml unlabeled anti-TCR antibody(Pharmingen), prior to the incubation with the liposomes. For inhibitionthrough partial blocking of MHC, the liposomes were incubated withunlabeled anti-MHC at a molar ratio of 1:1 to incorporated MHC. Forcompetition/inhibition with non-biotinylated peptide, non-biotinylatedpeptide was added together with the biotinylated peptide, and at thesame molar ratio to the MHC.

EXAMPLE 17 Characterization of Artificial APCs

Mean particle sizes of the MHC-containing liposomes were determinedthrough dynamic light scattering analysis with a Malvern 4700 system,using a 25 mW Ne—He laser and the automeasure version 3.2 software(Malvern, Ltd.). For refractive index and viscosity the values for purewater were used. The particle size distribution was reflected in thepolydispersity index (p.i.:0, which ranges from 0.0 for a monodisperseto 1.0 for a polydisperse dispersion). Besides DLS analysis, liposomeswere visualized through flow cytometric analysis as described beforeherein. Briefly, FITC-labeled liposomes were gated according to theirFL1 fluorescence by placing a threshold at the FL1 channel, while forforward and side scattering the most sensitive settings were selected.The size of the FITC-liposomes was compared with the FSC of 2 types ofFITC-labeled calibration beads ranging in sizes from 40 to 60 nm andfrom 700 to 900 nm (Pharmingen). The results are shown in FIG. 20wherein the shaded area shows that the artificial APCs size peak is inthe range of 0.05 μm.

For qualitative analysis of the MHC class II incorporation efficiency asexplained in Example 16, MHC class-II liposomes were incubated withPE-labelled anti-MHC class II mAbs (anti-DR4 (Parmogen); anti-I-Ad(clone 0X-6, Harlan)) or PE-labelled isotype controls and analyzed forPE-staining on the FACS calibur. MHC incorporation was quantitativelyanalyzed by a Petterson modification of the Lowry protein assay.Briefly, a standard curve (0–1–2–4–6–8–12 μg of MHC) was made with thesame detergent solubilized MHC batch as used for the preparation of theartificial APC. A reliable range for protein determination was between2–12 mg of MHC. All samples were analyzed in duplicate and linearregression was performed on the standard curve to evaluate the amount ofprotein incorporated in the liposome preparation. To calculate thenumber of MHC molecules per liposome, the amount of MHC, as determinedby the Petterson modified Lowry method, and the size of the liposomes,as determined by DLS, were used to convert the amount of incorporatedMHC into the number of MHC per liposome, assuming that a 100 nm liposomecontains approximately 80,000 phospholipid molecules/vesicle, andassuming that the average area per phospholipid molecule is 75 μm².

For qualitative analysis of peptide loaded MHC class II molecules, MHCmolecules were preloaded with biotinylated-peptide using the optimizedpeptide-MHC loading conditions. After MHC incorporation, the artificialAPC were incubated with Streptavidin-CY (Pharmingen), and analyzed forFL3 staining using the FACS calibur. The quantitative analysis ofoccupancy of the number of MHC molecules with the desired peptide wasperformed in 2 different ways, namely, soluble ELISA and by the analysisof unbound biotinylated peptide after SDS-PAGE and blotting of MHCmolecules incubated with peptide.

In addition to the MHC samples incubated with a dose range ofbiotinylated peptide, the same dose range of biotinylated peptide wasincubated under exactly the same experimental conditions without theaddition of MHC. After 24 hrs., two gels were run under identicalconditions in one minigel-system (Biorad). One gel was loaded with theMHC-peptide samples, while the other gel was loaded with the peptidesamples. The fronts of the gels were scanned by using the MolecularAnalyst software (Maxsott), and a standard curve was made by linearregression of the peptide loaded gel. After front analysis of theMHC-peptide gel the decrease of the peptide signal was quantified bycomparison with the standard curve and consequently, the amount of MRCbound peptide could be determined, (see FIG. 19).

Methods

Peptide Loading of MHC Class II Molecules

To determine optimal loading conditions for MHC class II molecules a MHCclass II binding assay was performed, essentially as described beforewith minor modifications. Briefly, detergent solubilized MHC class IImolecules (0.5–1 μM) were incubated with a 50–250-fold molar excess ofbiotinylated peptide for the designated time, pH and temperature withoutthe addition of protease inhibitors. The following conditions weretested; pH 5 and pH 7; Room Temperature and 37° C.; 16, 24 and 40 hoursof peptide loading; 0.05-, 1-, 10-, 100-fold molar excess of peptide.For DRB1*0401 0.5 μM, for I-Ad 3 μM of MHC was used in binding assays.

MHC-peptide complexes were analyzed via a non-reducingSDS-polyacrylamide gel electrophoresis (SDS-PAGE). Following WesternBlotting (Highland-ECL, Amersham), the biotinylated peptides werevisualized on preflashed films (Hyperfilm, Amersham) through enhancedchemiluminescence (Western blot ECL kit, Amersham). Optimal bindingconditions were established by evaluation of the density of the spots atthe position of the MHC class II dimer-peptide complexes. The presenceof the MHC Class II dimer under the tested conditions was checked with anon-reducing SDS-PAGE followed by silver staining (Biorad). (see FIGS.19A–D)

Preparation of Liposomes

Liposomes were prepared as follows. Stock solutions of egg PhosphatidylCholine (Sigma) and Cholesterol (Sigma) were combined in a glass tube ata molar ratio of 7:2.N-(fluorescein-5-thiocarbamoyl)-1,2-dihexadecanoyl-sn-glyvero-3-phosphoethanolamine,triethylammonium salt (fluorescein-DHPE, Molecular Probes, Eugene,Oreg.) was added at a final concentration of 1 mol % of PhosphatidylCholine. The solvent was evaporated under an Argon stream for 30 minutesand the lipids were dissolved at a final concentration of 10 mg/ml in abuffer containing 140 mM Nad, 10 mM Tris HCl (at pH 8) and 0.05%deoxycholate (Buffer A). The solution was sonicated until clear, andaliquoted in 500 μl Eppendorf tubes and stored at minus 200° C.

Incorporation of MHC-peptide in Liposomes

After preincubation of biotinylated peptides and MHC at conditions basedon in vitro MHC binding, MHC-peptide complexes were added to the lipidsin Buffer A at a ratio (w/w) of MHC:liposomes of 1:7 (DR4) or 1:10 (1Adand 1Ed). Additionally, for each T cell capture sample a ratio of 1 μgMHC per 6000 cells was used.

The lipids and MHC-peptide complexes were dialyzed at 4° C. against PBSin a 10K Slide A Lyzer (Pierce) for 48 hours with two buffer changes.The formed liposomes were then recovered from the Slide A lyzers andincubated with streptavidin-CY for 20 minutes before adding theliposomes to the cells. For MHC staining, liposomes were stained with aPE-labeled anti-MHC antibody at the same time.

Where other molecules of interest are to be incorporated into theartificial APC (i.e., accessory, adhesion, co-stimulatory, cellmodulation, irrelevant, GM-1, and cholesterol molecules), such moleculescan be added simultaneously with the incorporation of the MHC:antigencomplexes.

Quantification of HLA-DR and PADRE Peptide Bound to Liposomes

A modified ELISA was used to assess the amount of HLA-DR bound to theliposomes. Briefly, liposomes containing HLA DR4*0401 and biotinylatedPADRE peptide were formed as described above. The resulting liposomeswere counted and sorted by FACS on (FITC) fluorescence and forwardscatter. The sorted samples were then divided into two lots for HLA andpeptide quantification by ELISA.

DR Quantification

The sorted liposome sample was initially incubated with 10×M excess ofneutra-avidin (Pierce) to cap the biotin on the peptide. This wasfollowed by an extensive dialysis with a 300 k MWCO membrane (Pierce) toremove excess unbound neutra-avidin. The sample was then incubated withbiotinylated mouse anti-DR (Parmagen) at 1:1 molar ratio, which wasagain followed by extensive dialysis. The tagged complex was incubatedwith neutra-avidin-HRP at 1:20,000 and the excess dialyzed as before. Aknown amount of soluble DR pre-incubated with non-biotinylated PADREpeptide (standard) was incubated with the same antibody used fordetecting the DR in the liposomes. Multiple epitope recognition by theantibody assured retention of the complex during dialysis steps. Boththe sorted sample and the standard were then developed with TMB-HRPsubstrate and read at 450–650 nm.

PADRE Peptide Quantification

The sorted liposome sample was incubated with non-biotinylated anti-DR(Pharmagen) at a 1:1 molar ratio. The sample was treated and developedin the same manner as for DR quantification described above. The peptidewas quantified based on its biotin tag. Analysis of the data wasadjusted to background and to appropriate negative controls.

EXAMPLE 18 Methods for Orienting Molecules of Interest in an ArtificialAPC

We have shown that incorporation of molecules of interest into theartificial APC liposome matrix yields insertion of such molecules in anorientation wherein the active center of the molecules face outward onthe APC at a rate of about 50%. We have found that proper orientationcan be dramatically increased to over 90% by applying a mechanism todirect the insertion of the molecules. This mechanism uses GM-1pentasaccharide which is a transmembrane molecule and has an affinityfor binding the P subunit for cholera toxin. When the GM-1 is associatedwith the liposome membrane of the APC, it can be used to bind choleratoxin which in turn can be attached to the molecule of interest. Byattaching the cholera toxin to a point distal to the active site of themolecule of interest, we can direct the orientation of the molecule ofinterest such that the active site will be placed in the artificial APCin an orientation favorable to interaction with T cells and variousmolecules. In a preferred embodiment, proper orientation is obtainedusing a recombinant fusion between the molecule of interest and the Psubunit of cholera toxin the construction of which will be wellunderstood by those skilled in the art of making recombinant fusionproteins. The β subunit can also be connected to a molecule of interestusing a linker.

Methods

GM1 Pentasaccharide

The structure of GM-1 has been elucidated and shown to bepentasaccharide. The molecule is branched having terminal sugars,galactose and sialic acid (n-acetyl neuraminic acid), which exhibitsubstantial specific binding interactions with the β subunit of choleratoxin. A smaller contribution to binding is derived from the N-acetylgalactose residue of the molecule. This binding interaction is mediatedthrough hydrogen bonding. Each of the 5 identical binding sites areprimarily within a single monomer of the B-pentarner. GMI gangliosidemay be purchased from commercial sources (Sigma #G7641).

Cholera β Subunit (CTB)

Cholera toxin (mw=84 kda) is comprised of two subunits; α (mw=27 kda)and β (mw=11.6 kda). The amino acid sequence has been determined atabout 11,604 da. The primary structure of the β subunit, which isresponsible for binding of the toxin to the cell receptor gangliosideGM1, has been determined. Cholera toxin's ability to bind well to suchtransmembrane structures makes it very attractive anchor for membraneproteins (e.g. molecules of interest as disclosed herein). Since the βsubunit is primarily involved in the binding to the GM1 pentasaccharide,the α subunits are not necessary. The use therefore of only the βsubunit makes issues respecting toxicity less important with respect touse of the toxin protein subunits in therapeutic applications such asdrug delivery or transport and manipulation of T cells ex vivo.

The receptor binding site on the CTB is found to lie primarily within asingle β-subunit, with a solvent-mediated hydrogen bond involving thetwo terminal sugars of GM1 (galactose and sialic acid). The binding ofGM1 to cholera toxin thus resembles a 2-fingered grip. Cholera toxinβ-subunit may be purchased from commercial sources (Sigma #C9903 orC7771) or synthesized by recombinant methodology.

Linkers for Attaching Cholera Toxin to Molecules of Interest

Linkers may be used to attach the cholera toxin subunit to the moleculeof interest. In a preferred embodiment, a linker such asN-succinimidyl[3-(2-pyridyl) dithio) propionate (SPDP, Sigma Prod. No.P3415). NHS-esters such as this yield stable products upon reaction withprimary or secondary amines. Coupling is relatively efficient atphysiological pH. Accessible α-amine groups present on the N-termini ofproteins react with NHS-esters and form amides. In this regard, reactionwith side chains of amino acids can also occur. A covalent amide bond isformed when the NHS-ester cross-linking agent reacts with primaryamines, releasing N-hydroxysuccinimide.

In another preferred embodiment, pyridyl disulfides can be used to reactwith sulfhydryl groups to form a disulfide bond. Pyridine-2-thione isreleased as a by-product of this reaction. These reagents can be used ascross-linkers and to introduce sulfhydryl groups into proteins.Conjugates prepared using these reagents are cleavable.Pyrimidine-2-thione groups are released upon reaction with free -SHs andthe concentration can be determined by measuring the absorbance at 343nm (Molar extinction coefficient=8.08×10⁻³ M-1 cm-1).

Results

The GM1 is a transmembrane molecule which can be associated with the“raft” or freely mobile molecules of interest in the lipid membrane. GMIhas been used in studying movement and interactions of co-stimulatorymolecules through cross-linking the molecule with flourescinated-taggedcholera toxin. This cross linking can be carried out in several ways asshown in FIG. 27. For example, in FIG. 27A, a synthetic gene encoding amolecule of interest is cloned into a commercial vector such as ageneric expressions vector (for example, DES Expression Vector,Invitrogen) and expressed. The recombinant product can be purified andlinked to GM-1 that is in an artificial APC. In a similar fashion (FIG.27B) the molecule of interest can be cloned into an expression vector asa fusion protein with cholera toxin β subunit. The fusion product can bepurified and mixed with an artificial APC containing GM-1 where thecholera moiety will bind to the GM-1. Additionally, as shown in FIG.27C, the cholera toxin (whether natural or recombinant) can be attachedto a linker, such as N-succinimidyl[3-(2-pyridyl) dithio]propionate,either to a complete β subunit molecule or during synthesis of arecombinant toxin molecule, the product of which can be then mixed witha GM-1 containing artficial APC.

We have found that cross-linkers are useful as “flexible hinges” whereprotein molecules can be covalently linked to allow for intercellularinteraction with transmembrane proteins (e.g., B7-CD28, ICAM-1-LFA-1,MHCs, TCRs, etc.). Once the GM1 is incorporated into the liposome of theAPC, cholera toxin-conjugated surface proteins can then be cross-linked.The system containing properly oriented molecules of interest can thenbe tested through ELISA, WESTERN, and by FACS analysis.

Modifications and other embodiments of the invention will be apparent tothose skilled in the art to which this invention relates having thebenefit of the foregoing teachings, descriptions, and associateddrawings. The present invention is therefore not to be limited to thespecific embodiments disclosed but is to include modifications and otherembodiments which are within the scope of the appended claims. Allreferences are herein incorporated by reference.

What is claimed is:
 1. A method of identifying a T cell specific for anantigen of interest, comprising: a) contacting a biological samplecontaining T cells specific for an antigen of interest with anartificial antigen presenting cell that presents a peptide derived fromthe antigen of interest in order to form a complex comprised of a T cellspecific for the antigen of interest and an artificial antigenpresenting cell that presents the peptide derived from the antigen ofinterest, wherein the artificial antigen presenting cell comprises: i. aliposome comprising a lipid bilayer, wherein the lipid bilayer iscomprised of neutral phospholipids and cholesterol; ii. at least oneGM-1 ganglioside molecule disposed in the lipid bilayer; iii. a choleratoxin β subunit bound to one of the GM-1 ganglioside molecules; iv. anMHC molecule loaded with the peptide derived from the antigen ofinterest, wherein the antigen-loaded MHC molecule is bound to thecholera toxin β subunit; and v. an accessory molecule that can stabilizean interaction between a T cell receptor and the antigen-loaded MHCmolecule; and b) detecting the complex, if formed, thereby identifying aT cell specific for the antigen of interest.
 2. A method according toclaim 1 wherein the neutral phospholipids are phosphotidylcholine.
 3. Amethod according to claim 1 further comprising the step of isolatingfrom the complex the T cell specific for the antigen of interest.
 4. Amethod according to claim 3 further comprising the step ofcharacterizing a functional phenotype of the isolated T cells.
 5. Amethod according to claim 1 wherein the biological sample is selectedfrom the group consisting of whole blood, blood cells, blood plasma, andtissue.
 6. A method according to claim 1 wherein the peptide derivedfrom the antigen of interest is selected from the group consisting of apeptide derived from a recipient of a graft, a cancer cell-derivedpeptide, a peptide derived from an allergen, a donor-derived peptide,and a peptide derived by epitope mapping.
 7. A method according to claim1 wherein the artificial antigen presenting cell also comprises a label.8. A method according to claim 7 wherein the label is bound to amolecule of the artificial antigen presenting cell selected from thegroup consisting of a neutral phospholipid, cholesterol, a GM-1ganglioside molecule, a cholera toxin β subunit, an MHC molecule, thepeptide derived from the antigen of interest, and an accessory molecule.9. A method according to claim 7 wherein the label is selected from thegroup consisting of biotin, vancomycin, a fluorochrome, FITC, and aradiolabel.